Nucleic acids arrays and methods of use therefor

ABSTRACT

Compilations of nucleic acids, articles of manufacture which are surfaces comprising multiple blocks of arrays comprising such compilations, methods of use of the compilations and arrays for detection of chromosomal disorders, such as a chromosomal aneuploidies, deletions, amplifications, and diagnosis and prognosis of syndromes associated with a contiguous gene abnormality and kits are provided.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patentapplication Ser. No. 60/471,216 filed May 16, 2003, and is acontinuation-in-part of U.S. patent application Ser. No. 10/273,399filed Oct. 15, 2002 now U.S. Pat. No. 7,335,470, which claims thebenefit of U.S. provisional patent application Ser. No. 60/329,030 filedOct. 12, 2001, all of which are incorporated herein by reference intheir entireties.

TECHNICAL FIELD

This invention provides sets of nucleic acids, and articles ofmanufacture that are surfaces having multiple arrays, and methods forthe detection of chromosomal abnormalities, such as chromosomalaneuploidies, amplifications, deletions, and the like, and a diagnosisor prognosis of syndromes associated with a contiguous gene abnormality.Articles of manufacture are provided that have multiple arrays, eacharray providing identical blocks having a set of cloned nucleic acidsselected as associated with a chromosomal disorder, and having anotherset of cloned nucleic acids that are selected as not associated with aknown disorder.

BACKGROUND

Genomic DNA microarray based comparative genomic hybridization (CGH) hasthe potential to perform faster, more efficiently and cheaper thantraditional CGH methods, which rely on comparative hybridization onindividual metaphase chromosomes. Array-based CGH uses immobilizednucleic acids arranged as an array on a biochip or a microarrayplatform. The so-called array or chip CGH approach can provide DNAsequence copy number information across the entire genome in a single,timely, cost-effective and sensitive procedure. The resolution of chipCGH is primarily dependent upon the number, size and map positions ofthe DNA elements within the array. Bacterial artificial chromosomes, orBACs, can each accommodate on average about 150 kilobases (kb) of clonedgenomic DNA, and are often used in the production of the array.

Array CGH uses genomic DNA from cells of a series of samples to becompared, for example, a test sample and a reference sample (e.g., asample from cells ideally free of known chromosomal aberrations). Thetwo samples are labeled with different fluorescent dyes, and are mixedand co-hybridized to immobilized nucleic acids, e.g., BACs, or otherclones that contain a set of cloned genomic DNA fragments thatcollectively include a pre-determined portion of a genome or an entiregenome. The resulting co-hybridization produces a fluorescently labeledarray, and the extent of fluorescence of each of the dyes on each spotreflects competitive hybridization of sequences in the test andreference genomic DNAs to the homologous sequences within theimmobilized nucleic acids. Theoretically, the copy number ratio ofhomologous sequences in the test and reference genomic DNA samplesshould be directly proportional to the ratio of their respectivefluorescent signal intensities at discrete BACs within the array. Theversatility of the approach allows detection of constitutionalvariations in DNA copy number in clinical cytogenetic samples such asamniotic samples, chorionic villus samples (CVS), blood samples andtissue biopsies. It also allows detection of somatically acquiredgenomic changes in tumorigenically altered cells, for example, from bonemarrow, blood or solid tumor samples.

SUMMARY

A feature of the invention is a surface for identifying a chromosomaldisorder in a sample taken from a subject, the surface having aplurality of non-contiguous microarrays, such that each microarraycomprises a plurality of cloned genomic nucleic acids immobilized on thesurface at discrete and known spots, and each microarray comprises afirst set of spots having nucleic acids associated with the chromosomaldisorder and a second set of spots having control nucleic acids for thechromosome, the control nucleic acids not being associated with anychromosomal disorder on the chromosome. Further provided is acombination comprising this surface. A related embodiment of thecombination further has a cover for at least one of the plurality ofmicroarrays. In this combination, the cover functions to separate fluidabove at least one microarray from fluid above other microarrays of theplurality. Generally, the surface is planar, and the cover is planar orarcuate in cross section.

The surface material is selected from the group consisting of a metal,silicon, a polymer plastic, paper, ceramic, quartz, gallium arsenide,metal, metalloid, cellulose, celluose acetate, nitrocellulose, and aglass. For example, a glass microscope slide is exemplary for thismaterial. In embodiments in which the material is a plastic, examplarymaterials are nylon, polycarbonate, polyethylene, polystyrene, teflon,polypropylene, poly(4-methylbuene), polystyrene/latex, polymethacrylate,poly(ethylene terephthalate), rayon, polyvinylbutyrate, andpolyvinylidene difluoride.

The surface in one embodiment has nucleic acids associated with aplurality of chromosomes. For example, the plurality of chromosomescomprises the genome of the subject, although a portion of the genome isalso within the scope of the claims. Accordingly, the plurality ofchromosomes can comprise autosomes. Alternatively or additionally, theplurality of chromosomes comprises at least one sex chromome. Thus inone embodiment, the plurality of chromosomes further comprises an Xchromosome and a Y chromosome. In an alternate embodiment the surfacehas nucleic acids associated with a single chromosome.

In general, the surface of the microarray comprises a plurality ofcloned genomic nucleic acids associated with the chromosomal disorderand a plurality of cloned genomic nucleic acids that are not associatedwith a known chromosomal disorder, i.e., a plurality of control portionsof the chromosome. In one embodiment, the plurality of cloned nucleicacids associated with the chromosomal disorder are located on achromosome that is pre-selected by the user of the surface providedherein. Either or both of the chromosome of interest, or the disorder ofinterest, can be preselected. Alternatively, the plurality of clonednucleic acids associated with the chromosomal disorder are located on aplurality of chromosomes, for example, a subset of chromosomes in thegenome of the subject, or all of the chromosomes of the subject.Accordingly, both the plurality of control (i.e., backbone) portions andthe plurality of disorder-associated portions are distributed among theplurality of chromosomes of the subject.

In general, the microarray further contains a plurality of clonednucleic acids associated with a plurality of chromosomal disorders.Although embodiments of the microarray having cloned nucleic acidsassociated with a single disorder are within the scope of the invention,in other embodiments the plurality chromosomal disorders is at leastabout 5 chromosomal disorders, or is at least about 10 chromosomaldisorders, or is at least about 50 chromosomal disorders, or is at leastabout 100 or 200 chromosomal disorders. In general, the number ofchromosomal disorders is less than about 1,000, for example, themicroarray contains about 5 to about 1,000 chromosomal disorders, orabout 10 to about 300 chromosomal disorders, or about 20 to about 200chromosomal disorders, or about 40 to about 100 chromosomal disorders.An embodiment of the surface has microarrays having nucleic acidsassociated with each of about 40 chromosomal disorders. This is a usefulnumber of inherited disorders, since a large majority of inheritedchromosomal disorders falls within this group of disorders, and such asurface is valuable for diagnosis and prognosis of a set of mostcommonly inherited chromosomal disorders.

The microarrays herein may further have at least one calibration spot.For example, the calibration spot includes a mixture of the plurality ofcloned genomic nucleic acid not associated with known disorders, and sois a positive control for binding nucleic acids in a reference sample,and also in a test sample. In one embodiment a calibration spot hascloned genomic nucleic acid not associated with known disorders, andalso located on the chromosome closely linked to centromere. Exemplarycalibration spots of this embodiment can have centromere-linked nucleicacid for any one, or any plurality or all of the chromosomes of asubject organism. An alternative calibration spot has a nucleic acidfrom an unrelated source, for example, has DNA from a bacterium, anamphibian, or a piscean. Such calibration spots are used withcalibration samples, including bacterial, amphibian or piscean nucleicacid, to monitor hybridization efficiency. The calibration samples havea known amount of labeled nucleic acid capable of annealing to, orhybridization with, the calibration spot, hence the efficiency ofhybridization of the calibration sample to the calibration spot isuseful as a positive control, and for adjustment of the photomultiplierfor reading data from the arrays.

The surface in another embodiment has at least one fluid barrier locatedbetween at least two of the microarrays, such that the barrier functionsto separate hybridization fluid, which is a thin layer of fluid aboveeach microarray, from fluid above other microarrays of the plurality onthe surface. In examples of this embodiment, a barrier can be locatedbetween each of two microarrays; alternatively, two barriers can belocated between each microarray in a linear series of three microarrays.The surface barrier is in one embodiment an elevated structure ofhydrophobic composition contiguous with the surface; alternatively, thebarrier is a hydrophobic strip printed on the surface. An example of theelevated structure is a glass barrier; alternatively, the elevatedstructure is a hydrophobic polymer barrier. Exemplary hydrophobic stripare strips printed from solutions or suspensions of polyethylene,silicone, paraffin, or Teflon®. Conditions of the application of strips,such as temperature of the applied material, nature of solvent, printingapparatus, are adjusted for each material.

The invention features in other embodiments a set of cloned genomicnucleic acids comprising portions of nucleotide sequences of at leastone chromosome of a subject, the set including a first subset of clonednucleic acids associated with at least one chromosomal disorder, and asecond subset of control cloned nucleic acids not associated with knownchromosomal disorders. Generally, the set has a plurality of chromosomaldisorders, and the cloned nucleic acids are known to be located at siteson a plurality of chromosomes, for example, autosomes, and/or sexchromosomes. The subject can be a mammal, although the set is useful forany nucleic acid bearing life form such as a virus, a bacterium, afungus, a protozoan, an alga, a multicellular invertebrate, acold-blooded vertebrate such as a fish or amphibian, or any bird orreptile. In general, the subject is a human, however the method is alsouseful for mammals that are rodents, carnivores, ungulates, equines,bovines, caprines, farm animals, zoo animals, agricultural animals, andagricultural plants such as corn, wheat, sorghum, rice and soy.

An embodiment of the invention is a compilation or set of nucleic acids,or an array containing this set, or a surface having multiples ofarrays, the set having a plurality of chromosomal disorders each havinga substantial frequency in a human population. A substantial frequencyis at least one in about 10⁵ births, is at least one in about 10⁴births, is at least one in about 10³ births, or is at least one in about10² births. Examples of chromosomal disorders are inherited diseases dueto chromosomal deletions, insertions, inversions, translocations, andduplications.

In an alternative embodiment, chromosomal disorders are associated witha variety of cancers, and the sets of cloned genomic nucleic acids canbe associated with particular cancers, such as cancers of the prostate,the skin, lymphomas and leukemias, breast, pancreatic, liver, and brain,and lung. In an alternative embodiment, the set can contain a subset ofcloned nucleic acids commonly associated with various hazardous chemicalor physical exposures, to be used for detection of such exposure in asample obtained from a subject. The sample can be any tissue or bodilyfluid containing a source of a nucleic acid. Commonly used are cheekswabs and blood samples, however any tissue or fluid having a cellularcomponent is a source of nucleic acid for the uses and methods herein.

It is a finding of the examples herein that chromosomal disorders aregenerally associated with or are located near to ends of chromosome ortelomeres, and that portions of a chromosome closely linked tocentromeres are more commonly free of chromosomal disorders. Closelylinked means that the portion of the chromosome is located between thetip of the chromosome and the centromere, for example, and closer to thecentromer than to the tip, a distance which varies as measured either inMb or in CM according to the length of the chromosome. In a longchromosome such as chromosome 1, a centromere-linked portion can bewithin 10, 20, 50 or even 100 or 150 Mb from the centromere. In a smallchromosome, a centromere-linked portion can be within 10, 20, 30 or 40Mb from the centromere. Accordingly, an embodiment of the inventionherein, in addition to providing a set of nucleic acids associated withchromosomal disorders or a “first subset”, also provides a second subsetwhich are “backbone” or control cloned nucleic acids, i.e., portions ofone or more chromosomes that are closely linked to the centromere(previously referred to as a kinetochore in plants), i.e., the set ofcloned nucleic acids generally features a subset of control clonednucleic acids not associated with known chromosomal disorders that areportions of the chromosome as closely linked to the centromere aspossible.

The set can be a set of cloned nucleic acids associated with chromosomaldisorders such as: 1p36; Adrenal Hypoplasia Congenita; AlagilleSyndrome; Angelman Syndrome; Azospermia Factor A; Azospermia Factor B;Azospermia Factor C; Bruton Agammaglobulinemia Tyrosine Kinase;Beckwith-Wiedemann Syndrome; Charcot-Marie Tooth 1A; Cri-du-chatSyndrome; DiGeorge 1/VCF Syndrome; DiGeorge 2 (10p13); Down Syndrome;Duchenne Muscular Dystrophy; Fragile X syndrome; Glycerol KinaseDeficiency; Greig Syndrome (GLI3), Hereditary Neuropathy with Liabilityto Pressure Palsies; Hypoparathyroidism, Sensorineural Deafness andRenal Dysplasia; Kallman Syndrome; Langer-Giedion Syndrome (Ext1 andTrpsI); Miller-Dieker Syndrome; Potocki-Shaffer Syndrome (with MultipleExostoses 2); Neurofibromatosis 1; Pelizaeus-Merzbacher Disorder;Polycystic Kidney Disorder Type I; Prader-Willi Syndrome; Retinoblastoma1; Rubinstein-Taybi Syndrome; Saethre-Chotzen Syndrome; Sex determiningregion Y; Smith-Magenis Syndrome; Sotos Syndrome; Steroid SulfataseDeficiency; Trichorhinophalangeal Syndrome; Tuberous Sclerosis 1;Williams-Beuren Syndrome; Wilm's Tumor; WilmsTumor-Aniridia-Genitourinary anomalies-Mental retardation (WAGRSyndrome); and Wolf-Hirschhorn Syndrome.

Alternatively, the set of chromosomal disorders is at least one selectedfrom the group of: 1p36; Adrenal Hypoplasia Congenita; AlagilleSyndrome; Angelman Syndrome; Azospermia Factor A; Azospermia Factor B;Azospermia Factor C; Bruton Agammaglobulinemia Tyrosine Kinase;Beckwith-Wiedemann Syndrome; Charcot-Marie Tooth 1A; Cri-du-chatSyndrome; DiGeorge 1/VCF Syndrome; DiGeorge 2 (10p13); Down Syndrome;Duchenne Muscular Dystrophy; Fragile X syndrome; Glycerol KinaseDeficiency; Greig Syndrome (GLI3), Hereditary Neuropathy with Liabilityto Pressure Palsies; Hypoparathyroidism, Sensorineural Deafness andRenal Dysplasia; Kallman Syndrome; Langer-Giedion Syndrome (Ext1 andTrpsI); Miller-Dieker Syndrome; Potocki-Shaffer Syndrome (with MultipleExostoses 2); Neurofibromatosis 1; Pelizaeus-Merzbacher Disorder;Polycystic Kidney Disorder Type I; Prader-Willi Syndrome; Retinoblastoma1; Rubinstein-Taybi Syndrome; Saethre-Chotzen Syndrome; Sex determiningregion Y; Smith-Magenis Syndrome; Sotos Syndrome; Steroid SulfataseDeficiency; Trichorhinophalangeal Syndrome; Tuberous Sclerosis 1;Williams-Beuren Syndrome; Wilm's Tumor; WilmsTumor-Aniridia-Genitourinary anomalies-Mental retardation (WAGRSyndrome); and Wolf-Hirschhorn Syndrome.

Alternatively, the set of chromosomal disorders provided herein isrelated to cancer, and includes cloned nucleic acids associated withtumors or proliferative disorders, for example, from chromosome 8, inparticular from the long arm of chromosome 8, in particular thetelomeric end of the long arm of chromosome 8, and from chromosomes 9and 22, in particular, a translocation involving chromosomes 9 and 22associated with leukemias and forming the oncogene abl. Similar sets orcompilations of nucleic acids are envisioned for diagnosing andprognosing exposure to hazardous materials and/or to radiation in theenvironment. These sets or compilations, and the arrays, or surfacesincluding arrays such as surfaces having multiple copies of arrays, canbe used in conjunction with tester cells, for example, cells of a cellculture that is used to monitor the environment for mutagenic chemicalagents and ionizing radiation.

Also featured herein is a method of detecting a chromosomal disorder innucleic acid samples, the method including: providing a substrateincluding a surface having a plurality of non-contiguous arrays, eacharray comprising a plurality of cloned genomic nucleic acids immobilizedon the surface at discrete and known spots; contacting a first arraywith a first solution comprising a detectably labeled first nucleic acidmixture under conditions allowing hybridization between nucleic acids inthe first array to nucleci acids in the first mixture, and contacting asecond array with a second solution comprising a detectably labeledsecond nucleic acid mixture under the conditions allowing hybridization,such that the first and second solutions are not in contact; andanalyzing amounts of detectable label associated with each spot in thefirst and second arrays, thereby analyzing the samples to detect thechromosomal disorder in the nucleic acid samples. This method is also amethod of performing a plurality of nucleic acid hybridizations on asingle surface of a substrate.

In various embodiments of the method, the first and second nucleic acidmixtures include nucleic acid sequences from a test subject and areference subject. In a related embodiment, the first nucleic acidmixture has test subject nucleic acid detectably labeled with a firstfluorescent dye and reference nucleic acid detectably labeled with asecond fluorescent dye, and the second nucleic acid mixture has testsubject nucleic acid detectably labeled with the second fluorescent dyeand reference nucleic acid detectably labeled with the first fluorescentdye. The reference subject is a member of the same species from the testsubject and does not carry known chromosomal disorders. Alternatively,the reference subject is a member of a different species as the testsubject. In related embodiments, the second nucleic acid is at least onecloned portion of a chromosome. For example, the cloned portion is atleast one BAC clone, or is a plurality of BAC clones. Further, theplurality of cloned genomic nucleic acids immobilized in each arrayincludes at least one cloned portion of the genome associated with achromosomal disorder on a chromosome, and at least one cloned portion ofthe genome not associated with a chromosomal disorder on the chromosome.In certain embodiments, the first and second solutions further comprisea viscosity increasing solute. The method further includes, aftercontacting the first array and the second array with the solutions,disposing a cover on each of the first and second solutions.

A feature of the present invention is a method of analyzing genomicnucleic acid of a subject for the presence of a chroosomal disorder, themethod comprising: contacting a surface comprising a surface having afirst microarray and a second microarray with a first nucleic acidmixture and a second nucleic acid mixture, respectively, the microarraysbeing non-contiguous on the surface, wherein each microarray comprises aplurality of cloned nucleic acids immobilized on the surface at discreteand known spots; wherein the first mixture comprises test subjectnucleic acids linked to a first detectable label and reference nucleicacids linked to a second detectable label, and the first mixture isapplied to the first microarray; and the second mixture comprisesreference nucleic acids linked to the first detectable label and testnucleic acids linked to the second detectable label, wherein the firstmixture and the second mixture are separately contacted to the first andsecond microarrays, respectively; hybridizing the solution nucleic acidsto the microarray nucleic acids under suitable conditions; and,analyzing amounts of first and second labels bound to spots in each ofthe first and second microarrays, thereby analyzing the sample for thepresence of the chromosomal disorders.

Accordingly, the method further involves, after contacting the surfacewith each of the first mixture and the second mixture, separatelyapplying a cover to each of the first mixture and the second mixture. Ina related embodiment, the first and second mixtures further comprise aviscosity-increasing solute. For example, the viscosity-increasingsolute is selected from glycerol, polyethylene glycol, albumin, gelatin,and dextran, or any other macromolecule capable of increasing viscosityat low concentration, without affecting rate of nucleic acidhybridization. In the method, analyzing amounts further is measuring thefirst and second labels bound to spots in the microarrays using anautomated scanner. In a related embodiment, analyzing amounts further ismeasuring labels bound to spots in the microarrays using a laserscanner. Alternatively, using the automated scanner is using a CCD.

For specific embodiments of the method, the first and second microarrayscomprise a plurality of cloned portions of the genome associated with achromosomal disorder on a chromosome, and a plurality of cloned portionsof the genome not associated with a chromosomal disorder on thechromosome. In certain embodiments, the first and second microarraysfurther comprise at least one calibration spot. The calibration spotcomprises a mixture of the plurality of cloned genomic nucleic acid fromportions of chromosomes not associated with known disorders. Further,the calibration spot comprises cloned genomic nucleic acid from aportion of chromosomes not associated with known disorders and furtherhaving locations on the chromosomes closely linked to a centromere.Accordingly, analyzing the amounts is contrasting amount of label boundto the reference nucleic acid spots, with amount of label bound tocalibration spots. In alternative embodiments, the calibration spotcomprises nucleic acids from a species different than the test subject,and wherein positive control samples comprise predetermined quantitiesof complementary nucleic acid sequences capable of hybridizing to thecalibration spots, and analyzing the positive control data to determinecopy number of a gene in the test sample.

Another feature of the invention is a kit comprising a surface having amultiplicity of micro-arrays as described herein, a container, andinstructions for use. The kit can further include nucleic acid of areference subject. The kit can further include a first detectable labeland a second detectable label.

In one aspect, the stringent hybridization conditions comprisepost-hybridization washing conditions comprising: pre-warming thefollowing hybridization solutions at 50° C. in individual Petri dishes:2×SSC, 50% deionized formamide, 2×SSC, 0.1% NP-40, 0.2×SSC; soaking thearray (e.g., a slide) in 2×SSC, 0.5% SDS briefly at room temperature(RT), or alternatively, just 2×SSC can be used; transferring the array(slide) to the pre-warmed 2×SSC, 50% formamide; washing the slides byincubating in the shaking incubator at 50° C. for 20 minutes. In oneaspect, the post-hybridization washing conditions comprise repeating thewash using a pre-warmed 2×SSC, 0.1% NP-40. In one aspect, thepost-hybridization washing conditions comprise repeating the wash usinga pre-warmed 0.2×SSC for 10 minutes. In one aspect, thepost-hybridization washing conditions comprise rinsing the slides withdistilled deionized water. In one aspect, this last wash does not exceed10 seconds. In one aspect, the arrays (slides) are immediately driedunder forced air.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patent, and GenBank sequences and other references mentioned herein areincorporated by reference in their entirety. In the case of conflict,the present specification, including definitions, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is drawing of human chromosome 1, with the ideogram showing p,the short arm, at the top and q, the long arm, at the bottom, and thecentromere as a constriction. In FIGS. 1-24, loci for map positions ofchromosomal disorders for each chromosome are indicated, as are loci notassociated with known chromosomal disorders (leftward pointing arrows).The identifiers of BAC clones carrying nucleic acid sequences for eachlocus are shown in the first column of the table in the figure. Thesecond column indicates the cytological position on the short or longarm. The third column indicates the position on the chromosome inmegabases, from the p terminus at the top.

FIG. 2 is a drawing of human chromosome 2, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 3 is a drawing of human chromosome 3, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 4 is a drawing of human chromosome 4, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 5 is a drawing of human chromosome 5, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 6 is a drawing of human chromosome 6, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 7 is a drawing of human chromosome 7, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 8 is a drawing of human chromosome 8, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 9 is a drawing of human chromosome 9, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 10 is a drawing of human chromosome 10, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 11 is a drawing of human chromosome 11, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 12 is a drawing of human chromosome 22, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 13 is a drawing of human chromosome 13, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 14 is a drawing of human chromosome 14, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 15 is a drawing of human chromosome 15, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 16 is a drawing of human chromosome 16, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 17 is a drawing of human chromosome 17, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 18 is a drawing of human chromosome 18, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 19 is a drawing of human chromosome 19, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 20 is a drawing of human chromosome 20, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 21 is a drawing of human chromosome 12, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 22 is a drawing of human chromosome 22, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 23A is a drawing of human chromosome X, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIG. 23B is a table related to the drawing of human chromosome X in FIG.23A.

FIG. 24 is a drawing of human chromosome Y, with arms, loci, positionsand clones marked as indicated for FIG. 1.

FIGS. 25A-25F shows a representation of human chromosomes 1-6 obtainedby multiple label reversal (“dye swap”) analysis, of a Wolf-Hirschhornpatient as the test sample, and a reference sample of a subject notknown to have any chromosomal disorders, the test sample and referencesample each labeled with a fluorescent dye, and then mixed andhybridized to an array of telomere-linked markers associated withchromosomal disorders, for each chromosome. For the graph of eachchromosome, only chromosomes 1-6 being shown herein, plots were obtainedin which with distal short arm, the p terminus, was positioned on theleft, and the distal long arm, the q terminus on the right. Plots wereactual ratios generated using the SpectralWare program (SpectralGenomics, Inc., Houston, Tex.). A ratio of 1.0 would be theoreticallyobtained when equal quantities of binding of test sample and referencesample nucleic acids to the spot having the cloned nucleic acid areobtained as a result of comparing the amount of binding of each nucleicacid to each spot. For ratio plots, each of the two sets of dye-swapdata can indicate insertion or deletion of genetic material, as shownhere and in FIGS. 26A-26F. Divergence of what the software prints asopen squares connected by a solid line below 1.0 on the ordinate, andwhat the software prints as black circles connected by a solid linebelow 1.0, indicates a loss of genetic material at the locus on thechromosome of the immobilized cloned nucleic acid as indicated on theabscissa. Similarly, divergence of the solid line above the 1.0 ratioline and the double line below 1.0 indicates a presence of anamplification or insertion of nucleic acid of the test sample nucleicacid, at the locus in the chromosome corresponding to the cloned nucleicacid indicated on the abscissa.

FIGS. 26A-26F are representation of human chromosomes obtained bymultiple dye swap analysis, using telomere-linked cloned loci associatedwith chromosomal disorders, for each chromosome, and control loci thatare not associated with known chromosomal disorders. Otherdeterminations are as shown in FIGS. 25A-25F.

DETAILED DESCRIPTION OF EMBODIMENTS

Novel compilations, or sets (e.g., clone sets), libraries orcollections, of nucleic acids and articles of manufacture which aresurfaces, i.e., arrays, are shown in application PCT/US02/33044, WO03/091426A1 published 6 Nov. 2003, the entire contents of which areincorporated herein by reference. In one aspect, these compilations, orsets, libraries or collections, of nucleic acids and arrays are used inthe detection of a chromosomal disorder or abnormality, such as achromosomal aneuploidy (an abnormality involving a chromosome numberthat is not an exact multiple of the haploid number). They can also beused in the diagnosis or prognosis of a syndrome associated with acontiguous gene abnormality.

The invention in one embodiment provides compilations, or sets,libraries or collections, of nucleic acids and arrays and methods forthe detection of a chromosomal abnormality or a diagnosis or prognosisof a syndrome associated with a contiguous gene abnormality. These orsets of nucleic acids and/or arrays can be used for routine or directedgenetic screening of embryos, fetuses, children or adults. These sets ofnucleic acids and/or arrays can be used to aid in the diagnosis orprognosis of a syndrome, particularly when it is suspected that apatient may have symptoms associated with one or more chromosomalabnormalities, but those symptoms are not definitively diagnostic.Screening of individuals before symptoms appear will allow preventativeor prophylactic treatment regimes.

The invention provides methods for selecting genomic fragments, or clonesets (including, e.g., libraries, collections or compilations offragments or clones), that are effective as hybridization targets in thedetection of chromosomal disorder abnormalities, such as aneuploidies(i.e., abnormalities involving a chromosome number that is not an exactmultiple of the haploid number), amplification, deletions and the like.In one aspect, these libraries, collections or compilations of genomicfragments or clones are immobilized on articles of manufacture, e.g.,arrays. In one aspect, articles of manufacture, e.g., arrays, comprisingthese libraries, collections or compilations of genomic fragments orclones (e.g., clone sets) are used to perform comparative genomichybridization (CGH) to detect chromosomal aneuploidies.

The selection process comprises choosing a clone containing a specificregion of the chromosome that hybridizes only to a single locus. Theselection process can also comprise selection of chromosome fragments(e.g., a plurality of clones) each containing a portion of the genomecontaining at least 15% unique sequences, i.e., sequences that are notpresent in the other regions of the genome. Choice of a plurality ofclones each of which contains a non-overlapping portion of closelylinked portions of a chromosome increases extent of resolution of thetechnique.

In another aspect, the article of manufacture is a surface having one ora multiple of arrays or microarrays, each array comprising a pluralityof as few as about 10 and up to at least about 2500 chromosome fragments(e.g., clones) selected by this method, for example, as described below.Each array of about 10, 40, 125, 250 or more clones is provided in aplurality, i.e., multiple copies which are at least two non-contiguouscopies. The genomic clones can be BAC, PAC, MAC, plasmids, recombinantviruses or phagemids and/or cosmids and the like. In one aspect, theselected chromosome fragments (e.g., clones) are cross-linked(immobilized) to a solid surface, e.g., an article of manufacture suchas an array.

In one aspect, the selected chromosome fragments (e.g., clones) areimmobilized as described in U.S. Pat. No. 6,048,695, the methods thereinproducing covalent linkage of the nucleic acids to the surface, whileminimizing non-specific binding of labeled sample nucleic acid as mightotherwise arise using a derivatized surface.

The article of manufacture can be an array comprising the selectedchromosome fragments (e.g., clones) immobilized on a surface, forexample, a glass slide. In one aspect, the slide is hybridized withfluorescently labeled test and control target DNA. In one aspect, thelibraries, collections or compilations of fragments or clones of theinvention comprise from about 5 up to about 1,000 or 2,500, for example,about 40 different chromosome fragments (e.g., clones) selected by thismethod, for example, as described below. Further, each fragment ispresent in at least one copy in the array, for example, is present intwo copies or three copies.

In one aspect, the articles of manufacture, e.g., plurality arrays, eachcomprises a plurality of nucleic acids segments immobilized on asurface, for example, as an array, or “biochip.” In a typical array orarray-like format, each segment can be immobilized onto a discrete andknown area, or “spot,” on the array. Each “spot” comprises a segment ofgenomic nucleic acid associated with a chromosomal abnormality, acontiguous gene abnormality, a genetically linked disease or a syndrome.In one aspect, while there may be many nucleic acids moleculesimmobilized on a particular spot, there is only one specie orrepresentation of a genomic nucleic acid segment associated with achromosomal abnormality per spot.

A subset of the spots of the array of the invention includes a pluralityof genomic nucleic acid segments, each associated with a chromosomalabnormality. Another subset of the spots are genomic nucleic acids notassociated with any known chromosomal abnormalities. In alternativeembodiments, as noted above, varying subpopulations of array spotscomprise such genomic nucleic acid segments. In certain embodiments, aset of spots includes nucleic acid segments that serve as positiveand/or negative controls. In one aspect, the test samples comprisecalibration spots, for example, test and or reference samples are“spiked” with known types and amounts of nucleic acids, such asheterologous nucleic acids, to serve as positive and negative controls.

Also provided are kits comprising the compilations, or sets, librariesor collections of the invention, and/or arrays of the invention. In oneaspect, the compilations, or sets, libraries or collections of theinvention, and/or arrays of the invention comprise, or consist of atleast one, or, all, of the clones as set forth in Tables and Figuresherein. The kits can include instructions for use of the compilations,or sets, libraries or collections, of nucleic acids and/or arrays andpracticing the methods of the invention, and, for the convenience of thepractitioner, materials for extracting genomic DNA from a sample andpreparing that DNA, including labeling of the genomic nucleic acid. Inone aspect, the kits can also include labeled “wild type” or referencegenomic nucleic acid, e.g., human genomic nucleic acid that is providedto serve as a “wild type,” i.e., genomic nucleic acid from a subjectknown not to have any or substantially having no known chromosomalabnormalities and/or any known contiguous gene abnormalities. The “wildtype” genomic nucleic acid comprises a substantially complete genome;which is useful if the practitioner is performing a comparative genomichybridization (CGH). The reference nucleic acid can be a mixture ofgenomic nucleic acids from several subjects known not to havechromosomal abnormalities or disorders.

BAC clones containing insert DNA, e.g., human genomic DNA, are stored in25% glycerol, and are kept frozen, for example, at minus 80° C. (storedin a −80° C. freezer).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. As used herein, the following terms havethe meanings ascribed to them unless specified otherwise.

The terms “array” or “microarray” or “DNA array” or “nucleic acid array”or “chip” or “biochip” as used herein is a plurality of target elements,each target element comprising a defined amount of one or morebiological molecules, e.g., genomic nucleic acid segments, immobilizedon a defined location on a substrate surface; as described in furtherdetail, below. In an embodiment of the invention herein, a surface has aplurality, or multiple copies of the array in a non-contiguousarrangement, so that a plurality of hybridizations can be conducted onthe surface.

The term “aryl-substituted 4,4-difluoro-4-bora-3a, 4a-diaza-s-indacenedye” as used herein includes all “boron dipyrromethene difluoridefluorophore” or “BODIPY” dyes and “dipyrrometheneboron difluoride dyes”(see, e.g., U.S. Pat. No. 4,774,339), or equivalents, are a class offluorescent dyes commonly used to label nucleic acids for theirdetection when used in hybridization reactions; see, e.g., Chen (2000)J. Org Chem. 65:2900-2906: Chen (2000) J. Biochem. Biophys. Methods42:137-151. See also U.S. Pat. Nos. 6,060,324; 5,994,063; 5,614,386;5,248,782; 5,227,487; 5,187,288.

The terms “cyanine 5” or “Cy5™” and “cyanine 3” or “Cy3™” refer tofluorescent cyanine dyes produced by Amersham Pharmacia Biotech(Piscataway, N.J.) (Amersham Life Sciences, Arlington Heights, IL.), asdescribed in detail, below, or equivalents. See U.S. Pat. Nos.6,027,709; 5,714,386; 5,268,486; 5,151,507; 5,047,519. These dyes aretypically incorporated into nucleic acids in the form of5-amino-propargyl-2′-deoxycytidine 5′-triphosphate coupled to Cy5™ orCy3™.

The terms “fluorescent dye” and “fluorescent label” as used hereinincludes all known fluors, including rhodamine dyes (e.g.,tetramethylrhodamine, dibenzorhodamine, see, e.g., U.S. Pat. No.6,051,719); fluorescein dyes; “BODIPY” dyes and equivalents (e.g.,dipyrrometheneboron difluoride dyes, see, e.g., U.S. Pat. No.5,274,113); derivatives of 1-[isoindolyl]methylene-isoindole (see, e.g.,U.S. Pat. No. 5,433,896); and all equivalents. See also U.S. Pat. Nos.6,028,190; 5,188,934.

The terms “hybridizing specifically to” and “specific hybridization” and“selectively hybridize to,” as used herein refer to the binding,duplexing, or hybridizing of a nucleic acid molecule preferentially to aparticular nucleotide sequence under stringent conditions. The term“stringent conditions” refers to conditions under which one nucleic acidwill hybridize preferentially to second sequence (e.g., a sample genomicnucleic acid hybridizing to an immobilized nucleic acid probe in anarray), and to a lesser extent to, or not at all to, other sequences. A“stringent hybridization” and “stringent hybridization wash conditions”in the context of nucleic acid hybridization (e.g., as in array,Southern or Northern hybridizations) are sequence dependent, and aredifferent under different environmental parameters. Stringenthybridization conditions as used herein can include, e.g., hybridizationin a buffer comprising 50% formamide, 5×SSC, and 1% SDS at 42° C., orhybridization in a buffer comprising 5×SSC and 1% SDS at 65° C., bothwith a wash of 0.2×SSC and 0.1% SDS at 65° C. Exemplary stringenthybridization conditions can also include a hybridization in a buffer of40% formamide, 1 M NaCl, and 1% SDS at 37° C., and a wash in 1×SSC at45° C. Those of ordinary skill will readily recognize that alternativebut comparable hybridization and wash conditions can be utilized toprovide conditions of similar stringency.

The selection of a hybridization format is not critical, as is known inthe art, as it is the stringency of the wash conditions that set forththe conditions which are determinative as to whether a soluble, samplenucleic acid will specifically hybridize to an immobilized nucleic acid.Wash conditions can include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl and atemperature of at least about 72° C. for at least about 15 minutes; or,a salt concentration of about 0.2×SSC at a temperature of at least about50° C. or about 55° C. to about 60° C. for at least about 15 to about 20minutes; or, the hybridization complex is washed twice with a solutionwith a salt concentration of about 2×SSC containing 0.1% SDS at roomtemperature for 15 minutes and then washed twice by 0.1×SSC containing0.1% SDS at 68° C. for 15 minutes; or, equivalent conditions. Stringentconditions for washing can also be, e.g., 0.2 ×SSC/0.1% SDS at 42° C.See Sambrook, Ausubel, or Tijssen (cited herein) for detaileddescriptions of equivalent hybridization and wash conditions and forreagents and buffers, e.g., SSC buffers and equivalent reagents andconditions.

The phrase “labeled with a detectable composition” or “labeled with adetectable moiety” as used herein refers to a nucleic acid comprising adetectable composition, i.e., a label, as described in detail, below.The label can also be another biological molecule, as a nucleic acid,e.g., a nucleic acid in the form of a stem-loop structure as a“molecular beacon,” as described below. This includes incorporation oflabeled bases (or, bases which can bind to a detectable label) into thenucleic acid by, e.g., nick translation, random primer extension,amplification with degenerate primers, and the like. The label can bedetectable by any means, e.g., visual, spectroscopic, photochemical,biochemical, immunochemical, physical or chemical means. Examples ofsuitable fluorescent materials include umbelliferone, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; an example of aluminescent material includes luminol; examples of bioluminescentmaterials include luciferase, luciferin, and aequorin.

The term “nucleic acid” as used herein refers to a deoxyribonucleotideor ribonucleotide in either single- or double-stranded form. The termencompasses nucleic acids containing known analogues of naturalnucleotides. The term also encompasses nucleic-acid-like structures withsynthetic backbones. DNA backbone analogues provided by the inventioninclude phosphodiester, phosphorothioate, phosphorodithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, morpholinocarbarniate, and peptide nucleic acids (PNAs); see Oligonucleotides andAnalogues, a Practical Approach, edited by F. Eckstein, IRL Press atOxford University Press (1991); Antisense Strategies, Annals of the NewYork Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS1992); Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Researchand Applications (1993, CRC Press). PNAs contain non-ionic backbones,such as N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982; 5,684,148; seealso, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol.144:189-197. Other synthetic backbones encompassed by the term includemethyl-phosphonate linkages or alternating methylphosphonate andphosphodiester linkages (see, e.g., U.S. Pat. No. 5,962,674;Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonatelinkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) AntisenseNucleic Acid Drug Dev 6:153-156). The term nucleic acid is usedinterchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer,probe and amplification product.

The term “genomic DNA” or “genomic nucleic acid” includes nucleic acidisolated from a nucleus of one or more cells, and, includes nucleic acidderived from (e.g., isolated from, amplified from, cloned from,synthetic versions of) genomic DNA. The genomic DNA can be from anysource, as discussed in detail, below. The term “wild type genomicnucleic acid” means a sample of genomic nucleic acid having no known orsubstantially no known contiguous gene abnormalities.

The term “a sample comprising a nucleic acid” or “sample of nucleicacid” as used herein refers to a sample comprising a DNA or an RNA, ornucleic acid representative of DNA or RNA isolated from a naturalsource, in a form suitable for hybridization (e.g., as a soluble aqueoussolution) to another nucleic acid or polypeptide or combination thereof(e.g., immobilized probes). The nucleic acid may be isolated, cloned oramplified; it may be, e.g., genomic DNA, episomal DNA, mitochondrialDNA, mRNA, or cDNA; it may be a genomic segment that includes, e.g.,particular promoters, enhancers, coding sequences, and the like; it mayalso include restriction fragments, cDNA libraries or fragments thereof,etc. The nucleic acid sample may be extracted from particular cells,tissues or body fluids, or, can be from cell cultures, including celllines, or from preserved tissue sample, as described in detail, below.

As used herein, the terms “computer” and “processor” are used in theirbroadest general contexts and incorporate all such devices. The methodsof the invention can be practiced using any computer/processor and inconjunction with any known software or methodology. For example, acomputer/processor can be a conventional general-purpose digitalcomputer, e.g., a personal “workstation” computer, includingconventional elements such as microprocessor and data transfer bus. Thecomputer/processor can further include any form of memory elements, suchas dynamic random access memory, flash memory or the like, or massstorage such as magnetic disc optional storage.

Generating and Manipulating Nucleic Acids

Making and using the compilations, or sets, libraries or collections ofthe invention and/or arrays of the invention, and practicing the methodsof the invention may involve the isolation, synthesis, cloning,amplification, labeling and hybridization (e.g., CGH) of nucleic acids.The compilations, or sets, libraries or collections of the inventioncomprise nucleic acid segments. These nucleic acid segments and/orimmobilized nucleic acid on the array can be representative of genomicDNA, including defined parts of, or entire, chromosomes, or entiregenomes. Comparative genomic hybridization (CGH) reactions, see, e.g.,U.S. Pat. Nos. 5,830,645; 5,976,790, are discussed in further detail,below. Nucleic acid samples, the compilations, or sets, libraries orcollections of the invention (comprising nucleic acid segments), and, insome aspects, immobilized nucleic acids, can be labeled with adetectable moiety, e.g., a fluorescent dye(s) or equivalent. Forexample, a first sample can be labeled with a fluor and a second samplelabeled with a second dye (e.g., Cy3™ and Cy5™). In one aspect, eachsample nucleic acid is labeled with at least one different detectablemoiety, e.g., different fluorescent dyes, than those used to label theother samples of nucleic acids.

In some cases, the nucleic acids may be amplified using standardtechniques such as PCR. Amplification can also be used to subclone orlabel the nucleic acid prior to the hybridization. The sample and/or theimmobilized nucleic acid can be labeled, as described herein. The sampleor the probe on the array an be produced from and collectively can berepresentative of a source of nucleic acids from one or more particular(pre-selected) portions of, e.g., a collection of polymerase chainreaction (PCR) amplification products, substantially an entirechromosome or a chromosome fragment, or substantially an entire genome,e.g., as a collection of clones, e.g., BACs, PACs, YACs, and the like(see below). The array-immbolilized nucleic acid or genomic nucleic acidsample may be processed in some manner, e.g., by blocking or removal ofrepetitive nucleic acids or by enrichment with selected nucleic acids.

In one aspect, samples are applied to the immobilized probes (e.g., onthe array) and, after hybridization and washing, the location (e.g.,spots on the array) and amount of each dye are read. The compilations,or sets, libraries or collections, of nucleic acids or plurality ofimmobilized nucleic acid segments can be representative of any segmentof genomic nucleic acid associated with a chromosomal abnormality, acontiguous gene abnormality, a genetically linked disease or a syndrome;including, e.g., part of or all of a chromosome or genome. Thecompilations, or sets, libraries or collections, of nucleic acids orarray-immobilized nucleic acid can be in the form of cloned DNA, e.g.,YACs, BACs, PACs, and the like, as described herein. As is typical ofarray technology, in one aspect, each “spot” on the array has a knownsequence, e.g., a known segment of genome or other sequence. Theinvention can be practiced in conjunction with any method or protocol ordevice known in the art, which are well described in the scientific andpatent literature.

General Techniques

The nucleic acids used to practice this invention, whether RNA, cDNA,genomic DNA, vectors, viruses or hybrids thereof, may be isolated from avariety of sources, genetically engineered, amplified, and/orexpressed/generated recombinantly. Any recombinant expression system canbe used, including, in addition to bacterial cells, e.g., mammalian,yeast, insect or plant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:411-418;Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic AcidsRes. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380;Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol.68:90; Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066. Double stranded DNA fragments may thenbe obtained either by synthesizing the complementary strand andannealing the strands together under appropriate conditions, or byadding the complementary strand using DNA polymerase with a primersequence.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridization,G-banding, CGH, SKY, FISH and the like are well described in thescientific and patent literature, see, e.g., Sambrook, ed., MOLECULARCLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring HarborLaboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993).

Cloning of Genomic Nucleic Acids

The compilations, or sets, libraries or collections of the invention,and arrays of the invention, comprise nucleic acids, e.g., genomicnucleic acid segments. The nucleic acids used in the arrays,compilations and methods of the invention, e.g., those immobilized ontoarrays or used as samples, can be obtained and manipulated by cloninginto various vehicles. If necessary, genomic nucleic acid samples can bescreened and re-cloned or amplified from any source of genomic DNA.Thus, in various aspects, forms of genomic nucleic acid used in themethods of the invention (including arrays and samples) include genomicDNA, e.g., genomic libraries, contained in mammalian and humanartificial chromosomes, satellite artificial chromosomes, yeastartificial chromosomes, bacterial artificial chromosomes, P1 artificialchromosomes, recombinant vectors and viruses, plasmids, and the like.

Mammalian artificial chromosomes (MACs) and human artificial chromosomes(HAC) are, e.g., described in Ascenzioni (1997) Cancer Lett.118:135-142; Kuroiwa (2000) Nat. Biotechnol. 18:1086-1090; U.S. Pat.Nos. 5,288,625; 5,721,118; 6,025,155; 6,077,697). MACs can containinserts larger than 400 kilobase (Kb), see, e.g., Mejia (2001) Am. J.Hum. Genet. 69:315-326. Auriche (2001) EMBO Rep. 2:102-107, has built ahuman minichromosomes having a size of 5.5 kilobase.

Satellite artificial chromosomes, or, satellite DNA-based artificialchromosomes (SATACs), are, e.g., described in Warburton (1997) Nature386:553-555; Roush (1997) Science 276:38-39; Rosenfeld (1997) Nat.Genet. 15:333-335). SATACs can be made by induced de novo chromosomeformation in cells of different mammalian species; see, e.g., Hadlaczky(2001) Curr. Opin. Mol. Ther. 3:125-132; Csonka (2000) J. Cell Sci. 113( Pt 18):3207-3216.

Yeast artificial chromosomes (YACs) can also be used and typicallycontain inserts ranging in size from 80 to 700 kb. YACs have been usedfor many years for the stable propagation of genomic fragments of up toone million base pairs in size; see, e.g., U.S. Pat. Nos. 5,776,745;5,981,175; Feingold (1990) Proc. Natl. Acad. Sci. USA 87:8637-8641;Tucker (1997) Gene 199:25-30; Adam (1997) Plant J.11:1349-1358;Zeschnigk (1999) Nucleic Acids Res. 27:21.

Bacterial artificial chromosomes (BACs) are vectors that can containcloned inserted DNA of length 120 kb or greater, see, e.g., U.S. Pat.Nos. 5,874,259; 6,277,621; 6,183,957. BACs are based on E. coli F factorplasmid cloning vehicle systems, and are simple to manipulate and purifyin microgram quantities. Because BAC plasmids are maintained in vivo atone to two copies per E. coli cell, the problems of rearrangementobserved with YACs, which can also be employed in the present methods,are substantially reduced or even eliminated; see, e.g., Asakawa (1997)Gene 69-79; Cao (1999) Genome Res. 9:763-774.

P1 artificial chromosomes (PACs), bacteriophage P1-derived vectors are,e.g., described in Woon (1998) Genomics 50:306-316; Boren (1996) GenomeRes. 6:1123-1130; Ioannou (1994) Nature Genet. 6:84-89; Reid (1997)Genomics 43:366-375; Nothwang (1997) Genomics 41:370-378; Kern (1997)Biotechniques 23:120-124). P1 is a bacteriophage that infects E. colithat can contain 75 to 100 kb DNA inserts (see, e.g., Mejia (1997)Genome Res 7:179-186; Ioannou (1994) Nat Genet 6:84-89). PACs arescreened in much the same way as lambda libraries. See also Ashworth(1995) Analytical Biochem. 224:564-571; Gingrich (1996) Genomics32:65-74.

Other cloning vehicles can also be used, for example, recombinantviruses; cosmids, plasmids or cDNAs; see, e.g., U.S. Pat. Nos.5,501,979; 5,288,641; 5,266,489.

These vectors can include marker genes, such as, e.g., luciferase andgreen fluorescent protein genes (see, e.g., Baker (1997) Nucleic AcidsRes 25:1950-1956). Sequences, inserts, clones, vectors and the like canbe isolated from natural sources, obtained from such sources as ATCC orGenBank libraries or commercial sources, or prepared by synthetic orrecombinant methods.

Amplification of Nucleic Acids

Amplification using oligonucleotide primers can be used to generate ormanipulate, e.g., subclone, nucleic acids of the compilations, or sets,libraries or collections of the invention, nucleic acids used in thearrays of the invention and for practicing the methods of the invention,or to incorporate label into immobilized or sample nucleic acids, or todetect or measure levels of nucleic acids hybridized to an array, andthe like. Amplification, typically with degenerate primers, is alsouseful for incorporating detectable probes (e.g., Cy5™- or Cy3™-cytosineconjugates) into nucleic acids representative of test or control genomicDNA to be used to hybridize to immobilized genomic DNA. Amplificationcan be used to quantify the amount of nucleic acid is in a sample, see,e.g., U.S. Pat. No. 6,294,338. The skilled artisan can select and designsuitable oligonucleotide amplification primers. Amplification methodsare also well known in the art, and include, e.g., polymerase chainreaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed.Innis, Academic Press, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis,Academic Press, Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu(1989) Genomics 4:560; Landegren (1988) Science 241:1077; Barringer(1990) Gene 89:117); transcription amplification (see, e.g., Kwoh (1989)Proc. Natl. Acad. Sci. USA 86:1173); and, self-sustained sequencereplication (see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA87:1874); Q Beta replicase amplification (see, e.g., Smith (1997) J.Clin. Microbiol. 35:1477-1491), automated Q-beta replicase amplificationassay (see, e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and otherRNA polymerase mediated techniques, e.g., nucleic acid sequence basedamplification, or, “NASBA,” see, e.g., Birch (2001) Lett. Appl.Microbiol. 33:296-301; Greijer (2001) J. Virol. Methods 96:133-147. Seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202.

Hybridizing Nucleic Acids

In practicing the methods of the invention, samples of nucleic acid,e.g., isolated, cloned or amplified genomic nucleic acid, are hybridizedto the compilations, or sets, libraries or collections of the inventionor arrays of the invention, including immobilized nucleic acids. Inalternative aspects, the hybridization and/or wash conditions arecarried out under moderate to stringent conditions. The inventionprovides methods for selecting a genomic nucleic acid segment for use asa hybridization target in a hybridization reaction, e.g., a comparativegenomic hybridization (CGH) reaction, for the detection of a chromosomalabnormality comprising, inter alia, selecting a chromosomal segment thathybridizes to a single locus under stringent conditions. Exemplaryhybridization conditions, including stringent hybridization conditions,are set forth below.

An extensive guide to the hybridization of nucleic acids is found in,e.g., Sambrook Ausubel, Tijssen. Stringent hybridization and washconditions can be selected to be about 5° C. lower than the thermalmelting point (T_(m)) for the specific sequence at a defined ionicstrength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence hybridizes to aperfectly matched probe. Very stringent conditions are selected to beequal to the T_(m) for a particular probe.

Exemplary stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 complementaryresidues on an array can comprise 42° C. using standard hybridizationsolutions (see, e.g., Sambrook), with the hybridization being carriedout overnight. Exemplary highly stringent wash conditions can alsocomprise 0.15 M NaCl at 72° C. for about 15 minutes. Exemplary stringentwash conditions can also comprise a 0.2×SSC wash at 65° C. for 15minutes (see, e.g., Sambrook). In one aspect, a high stringency wash ispreceded by a medium or low stringency wash to remove background probesignal. An exemplary medium stringency wash for a duplex of, e.g., morethan 100 nucleotides, comprises 1×SSC at 45° C. for 15 minutes. Anexemplary low stringency wash for a duplex of, e.g., more than 100nucleotides, can comprise 4× to 6×SSC at 40° C. for 15 minutes.

In embodiments herein, in making the compilations, or sets, libraries orcollections, of nucleic acids or arrays, and practicing the methods ofthe invention, the fluorescent dyes Cy3™ and Cy5™ are used todifferentially label nucleic acid fragments from two samples, e.g.,nucleic acid generated from a control (e.g., “wild type” or referencenucleic acid), to be compared to a test cell or tissue sample (samplenucleic acid). In general, both the reference and sample nucleic acidsare labeled with each of Cy3™ and Cy5™, and two mixtures are made for a“dye swap” analysis. An embodiment of the methods and surfaces herein isthat in providing at least two copies of an array on a single surface,both mixtures of nucleic acids needed to perform the dye swap can behybridized to the same surface simultaneously. A variety of differenttechniques can be used alone or in combination to maintain separatefluids for the multiple hybridizations so that each sample is restrictedto one array on the surface. As described herein, a cover can be used;other techniques include use of a barrier which is a hydrophobic strip,or a raised barrier above the plane of the surface; and aviscosity-increasing solute can be used to decrease fluiditysufficiently to maintain the position of the fluid above the array. Acover can hold a predetermined volume of the fluid above the microarrayby surface tension, for example, a cover slip, or can physically impedemovement of the fluid.

Many commercial instruments are designed to accommodate the detection ofthese two dyes. To increase the stability of Cy5™, or fluors or otheroxidation-sensitive compounds, antioxidants and free radical scavengerscan be used in hybridization mixes, the hybridization and/or the washsolutions. Thus, Cy5™ signals are dramatically increased and longerhybridization times are possible.

In alternative aspects, the methods of the invention are carried out ina controlled, unsaturated humidity environment, and, the compilations,or sets, libraries or collections, of nucleic acids or arrays of theinvention can further comprise apparatus or devices capable ofcontrolling humidity. Controlling humidity is one parameter that can bemanipulated to increase hybridization sensitivity. Thus, in one aspect,in practicing the methods of the invention, hybridization can be carriedout in a controlled, unsaturated humidity environment; hybridizationefficiency is significantly improved if the humidity is not saturated.The hybridization efficiency can be improved if the humidity isdynamically controlled, i.e., if the humidity changes duringhybridization. Array devices comprising housings and controls that allowthe operator to control the humidity during pre-hybridization,hybridization, wash and/or detection stages can be used. The device canhave detection, control and memory components to allow pre-programmingof the humidity (and temperature and other parameters) during the entireprocedural cycle, including pre-hybridization, hybridization, wash anddetection steps.

In alternative aspects, the methods of the invention can incorporatehybridization conditions comprising temperature fluctuations and, thecompilations, or sets, libraries or collections, of nucleic acids orarrays of the invention can further comprise apparatus or devicescapable of controlling temperature, e.g., an oven. Hybridization hasmuch better efficiency in a changing temperature environment as comparedto conditions where the temperature is set precisely or at relativelyconstant level (e.g., plus or minus a couple of degrees, as with mostcommercial ovens). Reaction chamber temperatures can be fluctuatinglymodified by, e.g., an oven, or other device capable of creating changingtemperatures.

In alternative aspects, the methods of the invention can comprisehybridization conditions comprising osmotic fluctuations, and, thecompilations, or sets, libraries or collections, of nucleic acids orarrays of the invention can further comprise apparatus or devicescapable of controlling osmotic conditions, e.g., generate a e.g., asolute gradient. Hybridization efficiency (i.e., time to equilibrium)can also be enhanced by a hybridization environment that compriseschanging hyper-/hypo-tonicity, e.g., a solute gradient. A solutegradient is created in a device. For example, a low salt hybridizationsolution is placed on one side of the array hybridization chamber and ahigher salt buffer is placed on the other side to generate a solutegradient in the chamber.

Fragmentation and Digestion of Nucleic Acid

In practicing the methods of the invention, the compilations, or sets,libraries or collections of nucleic acids, the immobilized and/or samplenucleic acids can be cloned, labeled or immobilized in a variety oflengths. For example, in one aspect, the genomic nucleic acid segmentscan have a length smaller than about 200 bases. Use of labeled genomicDNA limited to this small size significantly improves the resolution ofthe molecular profile analysis, e.g., in array-based CGH. For example,use of such small fragments allows for significant suppression ofrepetitive sequences and other unwanted, “background”cross-hybridization on the immobilized nucleic acid. Suppression ofrepetitive sequence hybridization greatly increases the reliability ofthe detection of copy number differences (e.g., amplifications ordeletions) or detection of unique sequences.

The resultant fragment lengths can be modified by, e.g., treatment withDNase. Adjusting the ratio of DNase to DNA polymerase in a nicktranslation reaction changes the length of the digestion product.Standard nick translation kits typically generate 300 to 600 base pairfragments. If desired, the labeled nucleic acid can be furtherfragmented to segments below 200 bases, down to as low as about 25 to 30bases, random enzymatic digestion of the DNA is carried out, using,e.g., a DNA endonucleases, e.g., DNase (see, e.g., Herrera (1994) J.Mol. Biol. 236:405-411; Suck (1994) J. Mol. Recognit. 7:65-70), or, thetwo-base restriction endonuclease CviJI (see, e.g., Fitzgerald (1992)Nucleic Acids Res. 20:3753-3762) and standard protocols, see, e.g.,Sambrook, Ausubel, with or without other fragmentation procedures.

Other procedures can also be used to fragment genomic DNA, e.g.mechanical shearing, sonication (see, e.g., Deininger (1983) Anal.Biochem. 129:216-223), and the like (see, e.g., Sambrook, Ausubel,Tijssen). For example, one mechanical technique is based on point-sinkhydrodynamics that result when a DNA sample is forced through a smallhole by a syringe pump, see, e.g., Thorstenson (1998) Genome Res.8:848-855. See also, Oefner (1996) Nucleic Acids Res. 24:3879-3886;Ordahl (1976) Nucleic Acids Res. 3:2985-2999. Fragment size can beevaluated by a variety of techniques, including, e.g., sizingelectrophoresis, as by Siles (1997) J. Chromatogr. A. 771:319-329, thatanalyzed DNA fragmentation using a dynamic size-sieving polymer solutionin a capillary electrophoresis. Fragment sizes can also be determinedby, e.g., matrix-assisted laser desorption/ionization time-of-flightmass spectrometry, see, e.g., Chiu (2000) Nucleic Acids Res. 28:E31.

Syndromes Associated with a Contiguous Gene Abnormality

In one aspect, the invention provides compilations, or sets, librariesor collections of nucleic acids and arrays and methods for the detectionof a chromosomal abnormality or for the diagnosis or prognosis of asyndrome associated with a contiguous gene abnormality. Any set orcombination of genomic nucleic acid segments associated with achromosomal abnormality, a contiguous gene abnormality, a geneticallylinked disease or a syndrome, without limitation, can be used in makingand using the compilations, or sets, libraries or collections, ofnucleic acids or arrays and practicing the methods of the invention,including genomic nucleic acid segments described herein and genomicnucleic acid segments or other nucleic acids not specificallyexemplified herein. For example, the compilations, or sets, libraries orcollections of nucleic acids and/or arrays of the invention cancomprise, or consist of, at least one, or all of the clones set forth inTable 1. The compilations, or sets, libraries or collections, of nucleicacids or arrays and methods of the invention also can comprise genomicnucleic acid segments set forth in the literature, see, e.g., Charles R.Scriver, et al., (2000) “The Metabolic and Molecular Bases of InheritedDisease,” 8^(th) edition, New York, McGraw-Hill; Pat Gilbert (2000) “TheA-Z Reference Book of Syndromes and Inherited Disorders: A Manual forHealth, Social and Education Workers” 3 Ed edition, Stanley Thornes PubLtd.; Suzanne B. Cassidy, et al. (Ed), (2001) “Management of GeneticSyndromes,” Wiley-Liss.

The compilations, or sets, libraries or collections of the inventionand/or arrays and methods of the invention can be used for thedifferential diagnosis of genetically linked diseases or syndromes,formulating appropriate treatment plans and estimating a prognosis. Themethods of the invention can be used in situations where the causality,diagnosis, or prognosis (e.g., severity, metastatic potential) of apathology or condition is associated with one or more genetic defects,e.g., a syndrome caused by a contiguous chromosomal disorder or defect.

A “chromosomal disorder” as used herein means one due to mutations inthe genomic nucleic acid, primarily DNA, that are due to amplifications,deletions, inversions, translocations, and generally fall into thecategory of aneuploidy or deviation of the correct dosage of achromosome or a portion of a chromosome. Chromosomal disorders can be“congenital” or inherited, i.e., present at or before birth, or can besomatic, i.e., associated with a disease such as cancer or exposure tohazardous materials or radiation. Somatic chromosomal disorders arelikely to be characterized as mosaic, i.e., affecting a portion of cellsof a tissue or organism.

Considering for example the somatic chromosomal disorders in cancer,determining the presence of a contiguous gene defect can be helpful inpredicting or diagnosing and the prognosis of the cancer, classifying acancer or formulating a treatment plan or prognosis. For example,metastasis suppressor genes on human chromosomes for cutaneous melanoma,as well as a variety of other forms of human cancer, have been locatedon, e.g., 7q21-22, 7q31.2-32, 8p21-12, 10q11-22, 11p13-11.2, 12p11-q13,12q24-ter, and 17pter-q23 (see, e.g., Goldberg (2000) Am. J. Hum. Genet.67(2):417-431; Ichikawa (2000) Asian J. Androl. 2(3):167-171).Chromosomal abnormalities are common in prostate cancer, including butnot limited to, trisomy, hyperdiploidy and aneusomy of chromosomes 7 and17 (Cui et al., Cancer Genet Cytogenet 107: 51, 19998), amplificationsof 6p, 7q, 8q, 9q, and 16q (van Dekken et al., Lab Invest. 83: 789,2003), deletions of 3q, 6q, 8p, 10q, 13q, 16q, 17p and 20q (Matsuyama etal., Aktuel Urol 34: 247, 2003 and Prostate 54, 103, 2003).

Accordingly, the methods and arrays of the invention can be used forpredicting, diagnosing and the prognosis of cancers; chromosomal damagefrom exposure to hazardous chemical and physical agents and certaininherited conditions. As is well known to those of ordinary skill inoncology, certain chromosomal disorders are correlated with less severeor more severe prognostic outcomes. It is contemplated that as arraysherein become widely standard and data is accumulated for a variety ofcancers, additional correlations are made between chromosomal disordersand associated diagnoses and prognoses.

A list below of inherited chromosomal disorders is exemplary of thistype of disorder and not further limiting, nor are the methods,compilations, arrays, and surfaces herein limited to inheriteddisorders.

1p Deletion Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 1, locus 1p;36, and the syndromedetected is 1p Deletion Syndrome. Patients with deletion of band1p36.33, have had clinical findings of obesity and hyperphagia; and theoverlap of manifestations with Prader-Willi syndrome. See, e.g., Eugster(1997) Am. J. Med. Genet. 70(4):409-412. Patients with karyotypicabnormalities resulting in monosomy for a portion of 1p36.3 can havemicrocephaly, mental retardation, prominent forehead, deep-set eyes,depressed nasal bridge, flat midface, relative prognathism, and abnormalears. See, e.g., Reish (1995) Am. J. Med. Genet. 59(4):467-475.

3p Deletion Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 3, locus 3p25-pter, and the syndromedetected is 3p Deletion Syndrome. Chromosome 3p deletions are thought tobe involved in the pathogenesis of sporadic endocrine pancreatic tumors(EPTs); also, von Hippel-Lindau's disease (VHL gene at 3p25.5) has beenassociated with EPTs. Chromosome 3p deletion is frequently involved insolid human tumors. See, e.g., Barghorn (2001) J. Pathol.194(4):451-458. Allele loss in some regions of chromosome 3p has beendetected in primary breast tumors. See, e.g., Maitra (2001) Am. J.Pathol. 159(1):119-130.

3p Duplication Syndrome and “C Syndrome”

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 3, locus 3p21-pter, and the syndromedetected is 3p Duplication Syndrome. A partial trisomy of chromosome 3P,an inverted duplication 3p22-->3pter(dup(3)(pter-->p26::p22(p26::p26-->ter)), was found to be associatedwith psychomotor retardation and slight dysmorphism. A partial 3ptrisomy, a 3p/17p translocation: t(3;7)(p253;p133), was found to beassociated with mental retardation and poor speech development. See,e.g., Smeets (2001) Genet. Couns. 12(1):85-89. “C syndrome,” a multiplecongenital anomaly/mental retardation (MCA/MR) syndrome, was found to beassociated with a duplication of 3p. See, e.g., McGaughran (2000) Am. J.Med. Genet. 94(4):311-315.

Wolf-Hirschhorn Syndrome and Pitt-Rogers-Danks syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 4, locus 4p16.3, and the syndromedetected is Wolf-Hirschhorn Syndrome. Wolf-Hirschhorn syndrome (WHS) isa well-known congenital malformation syndrome caused by deletion of theshort arm of chromosome 4 (4p-). Most cases occur de novo and are ofpaternal origin. WHS children have severe developmental disabilities.The phenotype of adult WHS is in general similar to that of childhoodWHS. Growth retardation, microcephaly and mental retardation are therule in both adults and children. Facial dysmorphism also remainssimilar. The main difference lies in the absence of serious internal(cardiac) abnormalities in adult WHS. See, e.g., Battaglia (2001) Adv.Pediatr. 48:75-113; Marcelis (2001) Genet. Couns. 12:35-48. See, e.g.,Kant (1997) J. Med. Genet. 34(7):569-572. Pitt-Rogers-Danks syndrome hasalso been associated with deletions on chromosome 4p16.

4p Duplication Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 4, locus 4p15.2-16.1, and thesyndrome detected is 4p Duplication Syndrome. Duplications of the distalhalf of 4p give rise to the partial trisomy 4 syndrome, characterized bya “boxer” nose configuration and deep-set eyes. These signs are usuallyobserved even in cases of small terminal duplications. A “tandem”duplication of4p16.1p16.3 has been detected in association with a subtledeletion of 4p16.3pter on the same chromosome in a patient with the WHSphenotype. See, e.g., Zollino (1999) Am. J. Med. Genet. 82(5):371-375.

Cri du Chat Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 5, locus 5p 15.2-pter, and thesyndrome detected is Cri du Chat Syndrome. Most patients withcri-du-chat syndrome have a de novo deletion of the short arm ofchromosome 5 (5p). Patients show phenotypic and cytogenetic variability.Examples of deletions include: terminal-46,XX,del(5) (pter - - -p15.2:); interstitial-46,XX,del(5) (pter - - - p15.2::p13.3 - - -qter);46,XX,der(5)t(5;11) (p15;q25)mat. Clinically, younger patients canhave a typical high-pitched cry, psychomotor retardation, microcephaly,growth rate failure, and craniofacial abnormalities including roundface, hypertelorism, broad nasal bridge, downward slanting palpebralfissures, and micrognathia. See, e.g., Mainardi (2001) J. Med. Genet.38(3):151-158; Van Buggenhout (2000) Am. J. Med. Genet. 90(3):203-215.

Miller-Dieker Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 7, locus 7p13.3, and the syndromedetected is Miller-Dieker Syndrome. Trisomy 5p and Miller-Diekersyndromes frequently are the result of unbalanced segregations ofreciprocal translocations of chromosomes 5 and 17 with other autosomes.Miller-Dieker Syndrome has been associated with a breakpoint inchromosome 17p13. Miller-Dieker syndrome patients can present withmental retardation, postnatal growth deficiency, generalized muscularhypotonia, seizures, microcephaly, cortical atrophy, partial agenesis ofcorpus callosum, cerebral ventriculomegaly, facial anomalies. See, e.g.,Mutchinick (1999) Am. J. Med. Genet. 85(2):99-104; Pollin (1999) Am. J.Med. Genet. 85(4):369-375.

Williams Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 7, locus 7q11.23, and the syndromedetected is William's Syndrome. Williams syndrome is typically due to acontiguous gene deletion at 7q11.23, and has been associated with adistinctive facial appearance, cardiac abnormalities, infantilehypercalcemia, and growth and developmental retardation, including mildto severe mental retardation. For example, Williams syndrome was seen ina karyotype having microdeletions at 7q11.23 and 7q36 and additionalchromosomal material at 7q36. See, e.g., Donnai (2000) Am. J. Med.Genet. 97(2):164-171; Wouters (2001) Am. J. Med. Genet. 102(3):261-265.

Langer-Giedion Syndrome (LGS) or TRPS II

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 8, locus 8q24.1, and the syndromedetected is Langer-Giedion Syndrome (LGS) or tricho-rhino-phalangealsyndrome type II (TRPS II). It comprises the clinical features of twoautosomal dominant diseases, TRPS I, and a form of multiplecartilaginous exostoses caused by mutations in the EXT1 gene. Incontrast to TRPS I patients, most TRPS II patients have cytogeneticallyvisible deletions and are often mentally retarded. See, e.g., Hilton(2001) Genomics 71(2):192-199; Nardmann (1997) Hum. Genet.99(5):638-643. Other syndromes with contiguous deletions of chromosome8q include Cohen syndrome (8q22-q23), Klip-Feil syndrome (8q22.2),hereditary spastic paraplegia (8q24), and benign adult familialmyoclonic epilepsy (8q23.3-q24. 1).

Trichorhinophalangeal Syndrome (TRPS) or TRPS I

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 8, locus 8q24.1, and the syndromedetected is Trichorhinophalangeal Syndrome (TRPS) or TRPS I. TRPS Iindividuals typically have dysmorphic features and severe short stature.TRPS comprises a distinctive combination of hair, facial and bonyabnormalities with variable expression. The absence of generalizedshortness of all phalanges, metacarpals and metatarsals distinguish itfrom TRPS III, and absence of exostosis and mental retardation rule outTRPS II. See, e.g., George (1998) J. Eur. Acad. Dermatol. Venereol.11(1):66-68; Naselli (1998) Pediatr. Radiol. 28(11):851-855.

9p Deletion Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 9, locus 9p, e.g., locus 9p22-pter,and the syndrome detected is 9p Deletion Syndrome. This syndrome hasbeen associated with de novo deletions in the short arm of chromosome 9.Patients can have developmental delay/mental retardation, seizures andlearning disabilities. Mental retardation can be of variable degrees andthere can be a marked deficit in visuo-praxic and visuo-spatial skillsassociated with memory disturbance. See, e g., Chilosi (2001) Am. J.Med. Genet. 100(2):138-144. In contrast, cases of tetrasomy 9p areextremely rare; the principal clinical manifestations of this conditionare characteristic craniofacial abnormalities, generalized hypotonia andsevere mental retardation, see, e g., Kobayashi (2000) J.Craniomaxillofac. Surg. 28(3):165-170.

DiGeorge Syndrome II

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 10, locus 10p13-p14, and the syndromedetected is DiGeorge Syndrome II. This syndrome is characterized byneural-crest-related developmental defects. Partial monosomy 10p is arare chromosomal condition and a significant proportion of patients showfeatures of DiGeorge syndrome (DGS) and velocardiofacial syndrome(VCFS). One patient with DiGeorge syndrome (DGS) phenotype had anunbalanced translocation [45,XY,−10,−22,+der(10),t(10;22)(p13;q11)]resulting in monosomy of 10p13-pter and 22q11 -pter. See, e.g., Dasouki(1997) Am. J. Med. Genet. 73(1):72-75; Lichtner (2000) J. Med. Genet.37(1):33-37; Epstein (2001) Trends Genet. 17(10):S13-17.

WAGR Syndrome II

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 11, locus 11p13, and the syndromedetected is WAGR Syndrome. The Wilms' tumor-aniridia-genitalanomalies-mental retardation (WAGR) syndrome is associated with anincreased risk for developing Wilms' tumor. WAGR (Wilms' tumor,aniridia, genital anomalies, and mental retardation) syndrome anomalieshave been associated with balanced reciprocal 7;11 translocation and an11p13 breakpoint. See, e.g., Crolla (1997) J. Med. Genet. 34(3):207-212;Ariel (1996) Pediatr. Pathol. Lab. Med. 16(6):1013-1021.

Beckwith-Wiedemann Syndrome (BWS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 11, locus 11p15.5, and the syndromedetected is Beckwith-Wiedemann Syndrome. Beckwith-Wiedemann syndrome(BWS) is an imprinting disorder characterized by somatic overgrowth,congenital malformations, and predisposition to childhood tumors.Chromosome 11p15.5 have been reported to have an imprinted gene clusterof 1 Mb, which has been implicated in a wide variety of malignancies andBWS. See, e.g., Li (2001) Genomics 74(3):370-376; Horike (2000) Hum.Mol. Genet. 9(14):2075-2083.

Potocki-Shaffer Syndrome (Multiple Exostoses II Locus)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 11, locus 11p11.2, and the syndromedetected is Potocki-Shaffer Syndrome (Multiple Exostoses II Locus).Potocki-Shaffer Syndrome is caused by a proximal deletion in the shortarm of chromosome 11. Patients having the syndrome can have oval defectsof the parietal bones (parietal foramina). See, e.g., Wu (2000) Am. J.Hum. Genet. 67(5):1327-1332.

Angelman Syndrome (AS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 15, locus 15q12 or 15q13, and thesyndrome detected is Angelman Syndrome. It has been reported to becaused by the haploinsufficiency of the 15q11-q13 region, and, de novodeletions of chromosome 15q11-q13. It has also been reported thatAngelman syndrome can be caused by genetic abnormalities affecting thematernal copy of chromosome region 15q12. It has been observed thatextra copies of this same genomic region, in the form of inv-dup(15) orintra-chromosomal duplications, of maternal origin, are usuallyassociated with a severe neurological phenotype characterized bydevelopmental delay and untreatable seizures. See, e.g., Torrisi (2001)Am. J. Med. Genet. 106(2):125-128; Baumer (1999) Hum. Genet.105(6):598-602; Greger (1997) Am. J. Hum. Genet. 60(3):574-580.

Prader-Willi Syndrome (PWS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 15, locus 15q12, and the syndromedetected is Prader-Willi Syndrome (PWS). PWS is a neuroendocrinedisorder reported to be due to: a large paternally derived chromosomedeletion of 15q11q13, to maternal uniparental disomy (UPD), orimprinting mutation (IC). Severe learning disabilities (e.g.,attention-deficit hyperactivity disorder), dyslexia, and excessivedaytime sleepiness are common symptoms in PWS. See, e.g., Manni (2001)Clin. Neurophysiol. 112(5):800-805; Fernandez-Novoa (2001) Rev. Neurol.32(10):935-938.

Rubinstein-Taybi Syndrome (RTS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 16, locus distal 16p13.3, and thesyndrome detected is Rubinstein-Taybi Syndrome (RTS). RTS is amalformation syndrome characterized by facial abnormalities, broadthumbs, broad big toes, and mental retardation. In a subset of RTSpatients, microdeletions, translocafions, and inversions involvingchromosome band 16p13.3 can be detected. Immunodeficiency can be aprominent feature of this syndrome and may predispose these patients torecurrent infections. See, e.g., Petrij (2000) J. Med. Genet.37(3):168-176; Villella (2000) Arch. Dis. Child. 83(4):360-361.

Charcot-Marie-Tooth Disease Type 1A(CMT-1A)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 17, locus 17p12, and the syndromedetected is Charcot-Marie-Tooth Disease Type 1A(CMT-1A).Charcot-Marie-Tooth neuropathy type 1 (CMT1) is a geneticallyheterogeneous group of chronic demyelinating polyneuropathies with locimapping to chromosome 17 (CMT1A), chromosome 1 (CMT1B) and to anotherunknown autosome (CMT1C). CMT1A accounts for 70-90% of cases ofCharcot-Marie-Tooth Disease Type 1 and is most frequently caused by thetandem duplication of a 1.4-Mb genomic fragment on chromosome 17p12.Locus 17p12 is also associated with the peripheral neuropathies, such ashereditary neuropathy with liability to pressure palsies (HNPP) (seebelow). Some analyses have suggested that the syndrome is associatedwith de novo 17p11.2 duplication, paternal in origin, arising fromunequal crossing over due to homologous recombination between flankingrepeat gene clusters. X-linked dominant Charcot-Marie-Tooth (CMTX)disease is a motor and sensory neuropathy caused by mutations in theconnexin 32 (CX32) gene. See, e.g., Badano (2001) Clin. Chem.47(5):838-843; Potocki (2000) Nat. Genet. 24(1):84-87.

Hereditary Neuropathy (HNPP)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 17, locus 17p12, and the syndromedetected is Hereditary Neuropathy with Liability to Pressure Palsies(HNPP). HNPP is an autosomal dominant disorder that results in arecurrent, episodic demyelinating neuropathy. It also can becharacterized by reversible episodes of sensorimotor deficits afterneural compression injuries. Also known as tomaculous neuropathy, HNPPis further characterized ultrastructurally by multiple focal thickenings(tomacula) of peripheral myelin and has an autosomal dominantinheritance. HNPP is associated with a 1.5-Mb deletion in chromosome17p11.2-12 and results from reduced expression of the PMP22 gene. See,e.g., Mersiyanova (2000) Hum. Mutat.15(4):340-347; Chance (2001) Phys.Med. Rehabil. Clin. N. Am. 12(2):277-291; Lane (2001) J. Hand Surg. [Am]26(4):670-674.

Miller-Dieker Syndrome/Isolated Lissencephaly

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 17, locus 17p13.3, and the syndromedetected is Miller-Dieker Syndrome/Isolated Lissencephaly. TheMiller-Dieker syndrome (type I lissencephaly) is a neuronal migrationdisorder that is associated with microdeletions in the short arm ofchromosome 17, at locus 17p13.3. For example, one patient was found tohave a de novo balanced translocation with breakpoint at 8p11.23 and17p13.3. In contrast, neurofibromatosis type I (NF1) is an autosomaldominant condition associated with mutations in the long arm ofchromosome 17, and characterized by neurofibromas, cafe-au-lait spotsand axillary freckling. See, e.g., King (2000) Acta Neuropathol. (Berl)99(4):425-427; Honda (1998) Brain Dev. 20(3):190-192.

Smith-Magenis Syndrome (SMS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 17, locus 17p11.2, and the syndromedetected is Smith-Magenis Syndrome (SMS). SMS is a clinicallyrecognizable syndrome comprising multiple congenital anomalies andmental retardation. Its symptoms can include facial anomalies,brachydactyly, severe mental retardation, and self-injuring behavior.SMS is associated with a microdeletion (an interstitial deletion) of theshort arm of chromosome 17, locus 17p11.2. Interestingly, a patient witha del(l 7)(p11.2p12) karyotype displayed symptoms of both SMS andJoubert syndrome (JS), the later characterized by cerebellar vermishypoplasia, hypotonia, ataxic gait, developmental delay, and abnormalrespiratory pattern. A prenatal case of SMS found dysmorphic facialfeatures, tetralogy of Fallot, a thymic duct remnant, pancreatic isletcell hyperplasia, and abnormal lung fissuring. See, e.g., Juyal (1996)Am. J. Hum. Genet. 58(5):998-1007; Natacci (2000) Am. J. Med. Genet.95(5):467-472; Thomas (2000) Fetal Diagn. Ther. 15(6):335-337. SMSpatients have a phase shift of their circadian rhythm of melatonin witha paradoxical diurnal secretion of the hormone. See, e.g., De Leersnyder(2001) J. Med. Genet. 38(9):586-590; De Leersnyder (2001) J. Pediatr.139(1):111-116; Smith (1998) Am. J. Med. Genet. 81(2):186-191.

Alagille Syndrome (AGS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 20, locus 20p11.2-p12, and thesyndrome detected is Alagille Syndrome (AGS), also known asarteriohepatic dysplasia. Patients can have a deletion in chromosome20p, with 20p11.23-p12.2 as the area of minimal overlap. One AGS casehad a paracentric inversion (PAI) of chromosome 20p12.2p13. Locus20p11.2-p12 encodes a ligand for the Notch1 transmembrane receptor,which plays a key role in cell-to-cell signaling during differentiation.See, e.g., Yuan (1997) Acta Paediatr. Jpn 39(6):647-652; Hol (1995) Hum.Genet. 95(6):687-690.

Digeorge/Velocardiofacial Syndrome (VCFS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 22, locus 22q11.2, and the syndromedetected is Digeorge/Velocardiofacial Syndrome (VCFS). VCFS can resultfrom a microdeletion on chromosome 22, locus 22q11.2. VCFS is associatedwith a broad clinical spectrum characterized by multiple congenitalmalformations, including cleft palate and cardiac anomalies, thatfrequently overlaps the DiGeorge syndrome. Estimates suggest that the22q11.2 deletion occurs in approximately 1 in 4000 live births. Clinicalstudies indicate that more than 30% of children with VCFS will developschizophrenia. Velofacial hypoplasia (Sedlackova syndrome) andvelocardiofacial (Shprintzen) syndrome are also both associated with del22q11.2. See, e.g., Eliez (2001) Am. J. Psychiatry 158(3):447-453;Fokstuen (2001) Eur. J. Pediatr. 160(1):54-57; Duke (2000) Arch.Otolaryngol. Head Neck Surg. 126(9):1141-1145.

Adrenal Hypoplasia Congenita (AHC)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp21, and the syndromedetected is Adrenal Hypoplasia Congenita (AHC). AHC patients have adeletion on the short arm of the X chromosome, locus p21.1 to p22. 1.AHC is a developmental disorder of the human adrenal cortex and has beenproposed to be caused by deletion or mutation of the DAX-1 gene withinlocus p21.1 to p22.1; DAX-1 is a member of the nuclear hormone receptorsuperfamily. The Xp21 syndrome should be considered in any infant withadrenal insufficiency. Measurement of serum triglycerides and creatinekinase activity and karyotype screening tests will facilitate earlydiagnosis. See, e.g., Peter (1998) J. Clin. Endocrinol. Metab.83(8):2666-2674; Cole (1994) Clin. Chem. 40(11 Pt 1):2099-2103, and theGlycerol kinase deficiency (GKD) discussion, below.

Duchenne/Becker Muscular Dystrophy

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp21, and the syndromedetected is Duchenne/Becker Muscular Dystrophy. Cardiac abnormalities,cardiomyopathy and skeletal muscle weakness have been described infemale carriers of the Xp21 (Duchenne and Becker) muscular dystrophies.Duchenne and Becker dystrophies have been associated with the absence oraltered expression of dystrophin in cardiac and skeletal muscles. Theyare frequently complicated by cardiac hypertrophy and dilatedcardiomyopathy. See, e.g., Grain (2001) Neuromuscul. Disord.11(2):186-191; Crilley (2000) J. Am. Coll. Cardiol. 36(6):1953-1958, andthe Glycerol kinase deficiency (GKD) discussion, below.

Glycerol Kinase Deficiency (GKD)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp21, and the syndromedetected is Glycerol Kinase Deficiency (GKD). Glycerol kinase deficiency(GKD) is an X-linked recessive disorder having a deletion on the shortarm of the X chromosome, locus p21.1 to p22.1. There are two types. anisolated form and a complex form. The clinical and biochemical phenotypeof isolated GKD may vary from a life-threatening childhood metaboliccrisis to asymptomatic adult ‘pseudohypertriglyceridaemia’, resultingfrom hyperglycerolaemia. The complex GKD is an Xp21 contiguous genesyndrome involving the glycerol kinase locus together with the adrenalhypoplasia congenita (AHC) or Duchenne muscular dystrophy (DMD) loci orboth. Complex GKD patients can have an “hourglass” appearance of themiddle of the face; hypertelorism; rounded palpebral fissures;esotropia; wide, flattened earlobes; and a downtumed mouth. See, e.g.,Sjarif (2000) J. Inherit. Metab. Dis. 23(6):529-547; Scheuerle (1995) J.Pediatr. 126(5 Pt 1):764-767.

Pelizaeus-Merzbacher Disease (PMD)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp22, and the syndromedetected is Pelizaeus-Merzbacher Disease (PMD). PMD is an X-linkedrecessive dysmyelinating disorder of the central nervous system. Mostpatients have point mutations in exons of the proteolipid protein (PLP1)gene or duplication of a genomic region that includes the PLP1 gene, onlocus Xp22, on the short arm of the X chromosome. See, e.g., Hobson(2001) Hum. Mutat. 17(2):152; Hodes (2000) Am. J. Hum. Genet.67(1):14-22; Inoue (1999) Ann. Neurol. 45(5):624-632.

Steroid Sulfatase Deficiency

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp22.3, and the syndromedetected comprises steroid sulfatase deficiency. X chromosome deletionsin the Xp22.3 region can result in steroid sulfatase deficiency andX-linked ichthyosis. In one patient, an interstitial deletion in Xp22.3involved the Kallmann (KAL) gene, the steroid sulfatase (STS) gene and aputative mental retardation locus (MRX). X-linked ichthyosis (XLI) is aninborn error of metabolism due to steroid sulfatase (STS) deficiency.X-linked ichthyosis is a disorder of keratinization characterized by ageneralized desquamation of large, adherent, dark brown scales.Extracutaneous manifestations include corneal opacity andcryptorchidism. See, e.g., Weissortel (1998) Clin. Genet. 54(1):45-51;Santolaya-Forgas (1997) Fetal Diagn. Ther. 12(1):36-39; Valdes-Flores(2001) Am. J. Med. Genet. 102(2):146-148.

Abnormalities of the SRY Locus

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome Y, locus SRY locus/Yp, and thesyndrome detected comprises abnormalities of the SRY (sex-determiningregion on the Y chromosome) locus. SRY has been identified at bandYp11.31p11.32 in normal XY males and in woman with XY gonadaldysgenesis. SRY signals have also been identified on Xp22 in one XXmale. Ullrich-Turner syndrome (UTS) has been associated with Y fragmentsand gonadoblastomas. Thus, some clinicians have suggested that UTSpatients should be examined for Y chromosome material, and that positivecases should have their dysgenic gonads excised due to the high risk ofmalignancy. See, e.g., Kadandale (2000) Am. J. Med. Genet. 95(1):71-74;Damiani (1999) J. Pediatr. Endocrinol. Metab. 12(6):827-83 1; Kadandale(2000) Microb. Comp. Genomics 5(2):71-74.

Sex Reversal (DSS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp21, and the syndromedetected is Sex Reversal (DSS). The Xp21 locus contains the gene Ahch,also known as Dax1. Ahch encodes a transcription factor that has beenimplicated in sex determination and gonadal differentiation. Mutationsin human AHC cause X-linked, adrenal hypoplasia congenita (AHC) andhypogonadotropic hypogonadism (HH). Studies have found Xp duplicationsin patients with sex reversal, with female or ambiguous genitaliaoccurring in spite of an intact Yp or SRY gene. Five different exchangeshave been described two or more times: t(X;Y)(p21;q11), t(X;Y)(p22;p11),t(X;Y)(p22;q11-12), t(X;Y) (q22;q12), and t(X;Y)(q28;q12). See, e.g., Yu(1998) Nat. Genet. 20(4):353-7; Vasquez (1999) Genet. Couns.10(3):301-334.

Kallman's Disease or Kallmann's Syndrome (KS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp22.3, and the syndromedetected is Kallman's Disease or Kallmann's syndrome (KS). KS ischaracterized by hypogonadotrophic hypogonadism in association withanosmia or hyposmia. KS can be associated with X-linked ichthyosis (XLI)in a contiguous gene syndrome comprising a genetic defect in the Xp22.3region. KS has also been associated with olfactory neuroblastoma. See,e.g., Maya-Nunez (1999) Clin. Endocrinol. (Oxf) 50(2):157-162; Zappia(1992) J. Otolaryngol. 21(1):16-19.

17p11.2 Duplication Syndrome and Birt-Hogg-Dube Syndrome (BHD)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 17, locus 17p11.2, and the syndromedetected is 17p11.2 Duplication Syndrome. Duplication of locus 17p11.2may be associated with Birt-Hogg-Dube syndrome (BHD), an autosomaldominant neoplasia syndrome characterized mainly by benign skin tumors(e.g., benign tumors of the hair follicle), and to a lesser extent,renal tumors, lung cysts, and spontaneous pneumothorax. The gene for BHDmay associated with renal neoplasia and for lung and hair-follicledevelopmental defects. See, e.g., Schmidt (2001) Am. J. Hum. Genet.69(4):876-82; Khoo (2001) Oncogene 20(37):5239-5242.

Idiopathic Epilepsy and Paroxysmal Dyskinesia

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 16, pericentromeric region, and thesyndrome detected is idiopathic epilepsy and paroxysmal dyskinesia. Thisis a homogeneous syndrome of autosomal dominant infantile convulsionsand paroxysmal (dystonic) choreoathetosis (ICCA). Use of the arrays andmethods of the invention may be particularly useful because motormanifestations of epilepsy and of paroxysmal dyskinesia may be difficultto differentiate clinically. See, e.g., Guerrini (2001) Epilepsia 42Suppl 3:36-41.

Hirschsprung Disease Type 2 and Waardenburg Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 13, locus 13q22, and the syndromedetected is Hirschsprung disease, including Hirschsprung disease type 2,and Waardenburg syndrome. Hirschsprung disease is a developmentaldisorder resulting from the arrest of the craniocaudal migration ofenteric neurons from the neural crest along gastrointestinal segments ofvariable length. Waardenburg-Shah syndrome is an auditory pigmentarydisorder. Hirschsprung disease, malrotation, isochromia, a profoundsensorineural hearing loss, and several other anomalies were found in aninfant with an interstitial deletion of 13q, see, e.g., Shanske (2001)Am. J. Med. Genet. 102(3):231-236.

Branchio-Oto-Renal (BOR) Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising 8, locus 8q13.3, and the syndrome detected isbranchio-oto-renal (BOR) syndrome. Branchio-oto-renal (BOR) syndrome isan autosomal dominant disorder involving hearing loss, branchialdefects, ear pits and renal abnormalities. The arrays and methods of theinvention can be used to distinguish it from oto-facio-cervical (OFC)syndrome, which is clinically similar to BOR syndrome, with clinicalfeatures in addition to those of BOR syndrome. See, e.g., Rickard (2001)Hum. Genet. 108(5):398-403.

Smith-Magenis Syndrome (SMS)

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome 1, locus 7p 11.2, and the syndromedetected is Smith-Magenis syndrome (SMS). Smith-Magenis syndrome (SMS)is a multiple congenital anomaly/mental retardation (MCA/MR) syndromelink to a contiguous-gene deletion syndrome, involving chromosome 1 7p11.2,whose incidence is estimated to be 1:25,000 live births. SMS ischaracterized by a specific physical, behavioral and developmentalpattern. The main clinical features consist of a broad flat midface withbrachycefaly, broad nasal bridge, brachydactily, speech delay, hoarsedeep voice and peripheral neuropathy. See, e.g., Di Cicco (2001 Int. J.Pediatr. Otorhinolaryngol. 59(2):147-150.

Leri-Weill Syndrome

In one aspect, the compilations, or sets, libraries or collections, ofnucleic acids or arrays of the invention comprise a segment of genomicnucleic acid comprising chromosome X, locus Xp22.3, and the syndromedetected is Leri-Weill syndrome. Leri-Weill syndrome is characterized byshort stature (SHOX), chondrodysplasia punctata (ARSE), bilateralMadelung deformity and mental retardation. See, e.g., Spranger (1999)Am. J. Med. Genet. 83(5):367-371.

Chromosome abnormalities are common causes of congenital malformationsand spontaneous abortions. They include structural abnormalities,polyploidy, trisomy, and mosaicism. Very few autosomal trisomies surviveto birth, the three most common being those for chromosome 13, 18 and 21giving rise to the syndromes named Patau, Edward's and Down'srespectively (see, e.g., Moore (2000) Eur. J. Hum. Genet. 8:223-228).Thus, in alternative aspects, the arrays methods of the invention areused to diagnose Patau Syndrome, Edward's Syndrome and Down's Syndrome.See, e.g., Djalali (2000) Prenat. Diagn. 20:934-935. Table 1 shows a setof exemplary chromosomal disorders that can be diagnosed by thecompilations, or sets, libraries or collections, of nucleic acids,arrays and methods of the invention:

TABLE 1 Chromosome Loci Profiles of Contiguous Gene Syndromes Chromosomenumber Locus Syndrome  1 1p36 1p Deletion Syndrome  3 3p25 - pter 3pDeletion Syndrome  3 3p21 - pter 3p Duplication Syndrome  4 4p16.3Wolf-Hirschhorn Syndrome  4 4p15.2 - 16.1 4p Duplication Syndrome  55p15.2 - pter Cri du Chat Syndrome  7 7p13.3 Miller-Dieker Syndrome  77p11.23 William's Syndrome  8 8q24.1 Langer-Giedion Syndrome (LGS)  88q24.1 Trichorhinophalangeal Syndrome (TRPS)  9 9p, usually 9p DeletionSyndrome 9p22 - pter 10 10p13p14 DiGeorge Syndrome II 11 11p13 WAGRSyndrome 11 11p15.5 Beckwith-Wiedemann Syndrome 11 11p11.2Potocki-Shaffer Syndrome (Multiple Exostoses II Locus) 15 15q12 AngelmanSyndrome 15 15q12 Prader-Willi Syndrome 16 Distal 16p13.3Rubinstein-Taybi Syndrome 17 17p12 Charcot-Marie-Tooth Disease Type1A(CMT-1A) 17 17p12 Hereditary Neuropathy with Liability to PressurePalsies 17 17p13.3 Miller-Dieker Syndrome/Isolated Lissencephaly 1717p11.2 Smith-Magenis Syndrome 20 20p11.2p12 Alagille Syndrome 2222q11.2 (also Digeoege/Velocardiofacial Syndrome see 1-p13p14) X Xp21Adrenal Hypoplasia Congenita (AHC) X Xp21 Duchenne/Becker MuscularDystrophy X Xp21 Glycerol Kinase Deficiency X Xp22 Pelizaeus-MerzbacherDisease X Xp22.3 Steroid Sulfatase Deficiency Y SRY locus/YpAbnormalities of the SRY locus

The sets of nucleic acids, arrays and methods of the invention can alsobe used to detect aneuploidy of chromosomes 13, 18, 21, X, and Y fromgenomic DNA from newborn uncultured blood samples (see, e.g., Jalal(1997) Mayo Clin. Proc. 72:705-710). Chromosomal abnormalities have beenreported to occur in approximately 1%-2% of viable pregnancies studiedby chorionic villus sampling at 9-11 weeks of gestation. See, e.g.,Harrison (1993) Hum. Genet. 92:353-358.

In in vitro fertilization (IVF) programs, preimplantation geneticdiagnosis (PGD) of oocytes and embryos has become the technique ofchoice to select against abnormal embryos before embryo transfer. Thus,in alternative aspects, the compilations, or sets, libraries orcollections, of nucleic acids, arrays and methods of the invention areused for preimplantation genetic diagnosis and the diagnosis ofchromosomal abnormalities and structural abnormalities in oocytes andembryos. See, e.g., Fung (2001) J. Histochem. Cytochem. 49:797-798.Thus, in alternative aspects, the compilations, or sets, libraries orcollections, of nucleic acids, arrays and methods of the invention areused with chorionic villus sampling (CVS) and fetal karyotyping. See,e.g., Sanz (2001) Fetal Diagn. Ther. 16:95-97.

Genetic defects are frequent among transgenic animals produced bypronuclear microinjection. A successful method for the screening offounder animals for a chromosomal abnormality prior to mating wouldgreatly reduce the costs associated with the propagation of thetransgenic lines, and improve the efficiency of transgenic livestockproduction. Thus, in alternative aspects, the compilations, or sets,libraries or collections, of nucleic acids, arrays and methods of theinvention are used in the production of transgenic animals,particularly, the screening of founder animals for gene defects prior tomating. See, e.g., Ibanez (2001) Mol. Reprod. Dev. 58:166-172.

Comparative Genomic Hybridization (CGH)

In one aspect, sets of nucleic acids of the invention, and/or the arraysand methods of the invention incorporate array-based comparative genomichybridization (CGH) reactions to detect chromosomal abnormalities, e.g.,contiguous gene abnormalities, in cell populations, such as tissue,e.g., biopsy or body fluid samples. CGH is a molecular cytogeneticsapproach that can be used to detect regions in a genome undergoingquantitative changes, e.g., gains or losses of sequence or copy numbers.Analysis of genomes of tumor cells can detect a region or regions ofanomaly under going gains and/or losses.

CGH reactions compare the genetic composition of test versus controlssamples; e.g., whether a test sample of genomic DNA (e.g., from a cellpopulation suspected of having one or more subpopulations comprisingdifferent, or cumulative, genetic defects) has amplified or deleted ormutated segments, as compared to a “negative” control, e.g., “normal” or“wild type” genotype, or “positive” control, e.g., a known cancer cellor a cell with a known defect, e.g., a translocation or deletion oramplification or the like.

Making and using the compilations, or sets, libraries or collections, ofnucleic acids, arrays and practicing the methods of the invention canincorporate all known methods and means and variations thereof forcarrying out comparative genomic hybridization, see, e.g., U.S. Pat.Nos. 6,197,501; 6,159,685; 5,976,790; 5,965,362; 5,856,097; 5,830,645;5,721,098; 5,665,549; 5,635,351; and, Diago (2001) American J. ofPathol. May; 158(5):1623-1631; Theillet (2001) Bull. Cancer 88:261-268;Werner (2001) Pharmacogenomics 2:25-36; Jain (2000) Pharmacogenomics1:289-307.

Arrays or “BioChips”

The invention provides articles of manufacture, such as arrays,comprising the nucleic acid compilations, or sets, libraries orcollections of the invention. For example, in one aspect, inventionprovides an article of manufacture comprising, or consisting of, atleast one, or all, of the nucleic acid segments described in Table 1.Making and using the compilations, or sets, libraries or collections, ofnucleic acids, arrays and practicing the methods of the presentinvention can incorporate any known “array,” also referred to as a“microarray” or “DNA array” or “nucleic acid array” or “biochip,” orvariation thereof. Arrays are generically a plurality of “targetelements,” or “spots,” each target element comprising a defined amountof one or more biological molecules, e.g., polypeptides, nucleic acidmolecules, or probes, immobilized on a defined location on a substratesurface. Typically, the immobilized biological molecules are contactedwith a sample for specific binding, e.g., hybridization, betweenmolecules in the sample and the array. Immobilized nucleic acids cancontain sequences from specific messages (e.g., as cDNA libraries) orgenes (e.g., genomic libraries), including, e.g., substantially all or asubsection of a chromosome or substantially all of a genome, including ahuman genome. Other target elements can contain reference sequences,such as positive and negative controls, and the like. The targetelements of the arrays may be arranged on the substrate surface atdifferent sizes and different densities. Different target elements ofthe arrays can have the same molecular species, but, at differentamounts, densities, sizes, labeled or unlabeled, and the like. Thetarget element sizes and densities will depend upon a number of factors,such as the nature of the label (the immobilized molecule can also belabeled), the substrate support (it is solid, semi-solid, fibrous,capillary or porous), and the like. Each target element may comprisesubstantially the same nucleic acid sequences, or, a mixture of nucleicacids of different lengths and/or sequences. Thus, for example, a targetelement may contain more than one copy of a cloned piece of DNA, andeach copy may be broken into fragments of different lengths, asdescribed herein. The length and complexity of the nucleic acid fixedonto the array surface is not critical to the invention. The array cancomprise nucleic acids immobilized on any substrate, e.g., a solidsurface (e.g., nitrocellulose, glass, quartz, fused silica, plastics andthe like). See, e.g., U.S. Pat. No. 6,063,338 describing multi-wellplatforms comprising cycloolefin polymers if fluorescence is to bemeasured. Arrays used in the methods of the invention can comprisehousing comprising components for controlling humidity and temperatureduring the hybridization and wash reactions.

In making and using the compilations, or sets, libraries or collections,of nucleic acids, arrays and practicing the methods of the invention,known arrays and methods of making and using arrays can be incorporatedin whole or in part, or variations thereof, as described, for example,in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270;6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098;5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854;5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; seealso, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; seealso, e.g., Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997)Biotechniques 23:1087-1092; Kem (1997) Biotechniques 23:120-124;Solinas-Toldo (1997) Genes, Chromosomes & Cancer 20:399-407; Bowtell(1999) Nature Genetics Supp. 21:25-32. See also published U.S. patentapplications Nos. 20010018642; 20010019827; 20010016322; 20010014449;20010014448; 20010012537; 20010008765. The present invention can use anyknown array, e.g., GeneChips™, Affymetrix, Santa Clara, Calif.;SPECTRALCHIP™ Mouse BAC Arrays, SPECTRALCHIP™ Human BAC Arrays andCustom Arrays of Spectral Genomics, Houston, Tex., and theiraccompanying manufacturer's instructions.

In alternative embodiments, the compilations, or sets, libraries orcollections, of nucleic acids of the invention, and the articles ofmanufacture, such as arrays, of the invention, can comprise one, severalor all of the human genomic nucleic acid segments set forth below inFIGS. 1-24. These clones are identified by RP, GS, CTC or other CT clonenames; the descriptors for clones are in Nature 409:953-958 (2001),“Integration of cytogenetic landmarks into the draft sequence of thehuman genome.” The BAC Resource Consortium. A second column gives thecytological position of each as a marker on either the short arm, p, orlong arm, q, of the chromosome indicated, e.g. 8p23.3 is at the p armtelomere of chromosome 8, as shown in the ideogram of the chromosome inFIG. 8. The numbers in the third column of each Fig. indicate the linearposition of the cloned nucleic acid segment on the chromosome inmegabases (Mb). These figures show clones that represent all 24 humanchromosomes at a resolution of about or less than about 1 Mb resolution.The resolution is determined in part by the number of different clonedportions in a given length of a chromosome selected to be spotted on thearray, and the nature of the cloned portions, i.e., whether they areoverlapping or non-overlapping, and if non-overlapping, the extent ofthe gap between the cloned portions.

Substrate Surfaces

The nucleic acid compilations, or sets, libraries or collections of theinvention can be immobilized (directly or indirectly, covalently or byother means) to any substrate surface. The arrays of the invention canincorporate any substrate surface, e.g., a substrate means. Thesubstrate surfaces can be of a rigid, semi-rigid or flexible material.The substrate surfaces can be flat or planar, be shaped as wells, raisedregions, etched trenches, pores, beads, filaments, or the like.Substrates can be of any material upon which a nucleic acid (e.g., a“capture probe”) can be directly or indirectly bound. For example,suitable materials can include paper, glass (see, e.g., U.S. Pat. No.5,843,767), ceramics, quartz or other crystalline substrates (e.g.gallium arsenide), metals, metalloids, polacryloylmorpholide, variousplastics and plastic copolymers, Nylon™, Teflon™, polyethylene,polypropylene, poly(4-methylbutene), polystyrene, polystyrene/latex,polymethacrylate, poly(ethylene terephthalate), rayon, nylon, poly(vinylbutyrate), polyvinylidene difluoride (PVDF) (see, e.g., U.S. Pat. No.6,024,872), silicones (see, e.g., U.S. Pat. No. 6,096,817),polyformaldehyde (see, e.g., U.S. Pat. Nos. 4,355,153; 4,652,613),cellulose (see, e.g., U.S. Pat. No. 5,068,269), cellulose acetate (see,e.g., U.S. Pat. No. 6,048,457), nitrocellulose, various membranes andgels (e.g., silica aerogels, see, e.g., U.S. Pat. No. 5,795,557),paramagnetic or superparamagnetic microparticles (see, e.g., U.S. Pat.No. 5,939,261) and the like. Reactive functional groups can be, e.g.,hydroxyl, carboxyl, amino groups or the like. Silane (e.g., mono- anddihydroxyalkylsilanes, aminoalkyltrialkoxysilanes,3-aminopropyl-triethoxysilane, 3-aminopropyltrimethoxysilane) canprovide a hydroxyl functional group for reaction with an aminefunctional group.

Multi-Array Surfaces

Multi-array surfaces provided herein have on each surface a plurality ofcopies of the micro-array, i.e., micro-arrays (arrays) of biologicalmolecules, for example, nucleic acids. The term “multi-array surface” or“surface” as used herein means an article of manufacture having aplurality of micro-arrays applied to a side or a face of a substrate. Ingeneral the micro-arrays are printed or spotted or otherwise depositedon the face of the substrate, in an arrangement such that themicro-arrays are non-contiguous, i.e., the arrays are distal fromeachother on the surface, or are not in contact, compared to the size ofeach array and the spacings of the spots within each array.

A multi-array surface having a plurality of arrays is desirable for thefollowing procedures: hybridizations are conducted in duplicate ortriplicate on a single surface. Previous to the present invention,duplicate or triplicate or even a greater number of replicated spotshave been described that are present on a single surface, however allspots were exposed to hybridization of a single hybridization mixture.The hybridization mixture is a solution that typically contains anucleic acid sample from a test subject labeled with a fluorescent dye,or a mixture of two different samples of nucleic acids of differentorigins, each labeled with the same or a different dye. Thehybridization mixture is formed prior to hybridization with the spots ofthe array on the surface, for example, the mixture includes nucleicacids from test subject labeled with a first fluorescent dye and nucleicacids that are a reference sample, labeled with a different dye. Thereference sample can be nucleic acids from a normal individual of thesame species as the test subject, or can be nucleic acids of a differentspecies, or nucleic acids from a single BAC clone or from a mixture ofBAC clones. For BAC clones, NCBI maintains a human BAC resource, whichprovides genome-wide information concerning large-insert clones thatintegrate cytogenetic, radiation-hybrid, linkage, and sequence maps ofthe genome. See www.ncbi.nlm.nih.gov/genome/cyto/hrbc.shtml.

It is often desirable, in analyzing such data, to perform thehybridization in two different formats that reverse the fluorescentlabels, what is commonly described as a “label reversal”, “label swap”or “dye swap” analysis. In a dye swap analysis, at least two nucleicacid samples are to be compared, and at least two mixtures are made. Inthe first mixture, a first label such as a first fluorescent dye is usedto identify the reference nucleic acid probe, and a second label such asa second fluorescent dye is used to identify the test sample, and afterlabeling each, the mixture is made. Then the labels are reversed, i.e.,a second mixture is made in which the reference nucleic acid probecarries the second dye and the test sample carries the first dye. Eachof the two mixtures provides a reference for the purpose of plottingamounts of hybridization of each solution nucleic acid, reference andtest sample, to each of the immobilized cloned nucleic acids. Theresults are plotted as a function of the linear position of each of thecloned immobilized nucleic acids on a chromosome. Then a representationis made of a portion or of an entire chromosome, or of a plurality ofchromosomes, or of a complete set of chromosomes (autosomes with orwithout sex chromosomes), i.e., of the entire genome. Results obtainedfrom analyzing both sets of data are combined to reveal changes thatwould otherwise be undetectable if label reversal was not used. This isbecause small fluctuations from a ratio of 1.0 become statisticallysignificant when the dye swap data are plotted together, which might notbe significant if only a single mixture was used.

Further, it is often desirable to compare multiple test subjects withthe same reference sample. In any of these uses, multiple identicalarrays are necessary.

Prior to the methods and surfaces as described herein, it has beennecessary to conduct such analyses using a plurality of differentreplicas of the printed surfaces. For example, a dye swap analysis wasperformed with two mixtures, the first being a mixture of the testnucleic acid labeled with the first dye and mixed with the referencenucleic acid labeled with the second dye, and the second being the testnucleic acid labeled with the second dye mixed with the referencenucleic acid labeled with the first dye, the two mixtues then beinganalyzed using two different surfaces.

The use of separate hybridizations on different surfaces can be a sourceof variability, e.g., in efficiency of binding of spots to each surface,hybridization due to variability in conditions, minor variations inconcentration of each nucleic acid, variation in concentrations,different efficiencies in elution of non-specifically bound materialsdue to minor variations in washing procedures or solutions, at the timeof hybridization to each separate surface, or variations inphotomultiplier settings in a scanner used to visualize and evaluate thearray, after hybridization to each separate surface. Accordingly, thepresent surfaces provided herein address this problem in the priorcommon usage by having multi-arrays, which are a plurality of arrays ona single surface.

In a non-limiting example, two arrays are located at distal ends of aplanar substrate such as a standard glass microscope slide, howeveralternative shapes and sizes of substrates, and shapes and sizes ofarrays, are within the scope of surfaces, kits and methods envisionedherein. For example, a substrate may be a one inch by 3 inch microscopeslide, and may have a plurality of arrays such as two arrays, one ateither end, or four arrays in a linear arrangement. A larger substratesuch as a square slide may have four arrays, one in each corner, or ninearrays with three arrays on each side and one in the middle.

Further, barriers to maintain separation of fluids deposited on eacharray during hybridization may be used, the barriers being placedbetween each of the arrays, in addition to embodiments of the surface inthe absence of barriers, as described herein. The barriers are physical“dykes” or “dams” having a height above the plane of the substrate faceor surface, and such barriers include raised portions of the substrateas manufactured, or as added subsequently. Alternatively, the barriersmay be hydrophobic materials that are printed on the substrate toproduce a “strip” which can prevent the flow of an aqueous solution fromone array to another. The barriers can be added before or after printingor depositing the micro-arrays, to produce the multi-array surfaces.

The barriers are comprised of a material that is not soluble in aqueoussolution, and the material hydrophobic. Exemplary hydrophobic materialsfor barrier construction or printing include: polyethylene, silicone,paraffin, and Teflon®.

Hybridization using the “multi-array surfaces” having multiple arrays ona single surface of a single substrate, is conducted by adding thehybridization mixture to the array and protecting the hybridizationmixture with a cover to prevent loss of volume of solution byevaporation, and to confine each hybridization of a particular sample ormixture of samples, labeled with one or more dyes as described above, tothe appropriate micro-array. A pre-determined amount of hybridizationmixture is deposited above the array, such that addition of a cover, forexample placed directly on the fluid, yields a resulting thin layer offluid above the array in which the sample nucleic acids can hybridize tocomplementary sequences within the array. Hybridization for each arrayon the surface is conducted under a separate cover.

Conditions for hybridization can be modified, for example, thehybridization solution can be altered, to assure fluid separation of themultiple hybridizations on the surface. For example, viscosity of thehybridization fluid may be increased to reduce fluidity by adding one ormore solutes that do not interact with the nucleic acids during thehybridization. Exemplary solutes include small molecules that areviscuous liquids such as glycerol, and polymers of small molecules suchas sugars which is exemplified by dextrans and starches such as cornstarch, polymers of amino acids which are synthetic polypeptides ornaturally occurring proteins such as albumins and gelatins, andsynthetic polymers, for example, polyethylene glycol, or polyacrylamideor agarose, each at a concentration sufficient to increase viscositywithout significantly affecting mobility of the solute nucleic acids forinteraction and hybridization (annealing to form a double strandedcomplex) to the immobilized nucleic acids. The viscosity increasingsolute may be chemically modified to improve its properties, forexample, to render it resistant to digestion by extracellular enzymes ofbacteria and fungi. Solutions for hybridization may be stored withantibiotic or growth inhibiting materials to retard spoilage duringstorage.

The multi-array surfaces and methods herein are not limited toperformance of dye swap analyses. For example, a multi-array surfacehaving four or nine arrays can be used to analyze multiple samples, forexample, a plurality of members of a nuclear family, or multiplesiblings and a proband carrying a chromosomal disorder, which can now beanalyzed together on a single substrate having multiple micro-arrays,using separate hybridizations. Further, any multiple number of subjectscan be analyzed simultaneously on a single substrate, or any one subjectcan be analyzed in mixtures of different reference samples. Differentreference samples can be different species, different known mutations,or different predeteremined single BAC or mixtures of BAC clones.

Chromosomal Analysis using Calibration Spots and Disease-Negative Clones

Calibration spots that act as positive controls for hybridization of asample, and that are located within an array have been described (see,U.S. patent application 2003-0186250-A1, published Oct. 2, 2003, andincorporated herein in its entirety by reference). In embodiments of thesurfaces and methods provided herein, calibration spots may include asubset of cloned nucleic acids, for example, those clones of the humangenome carrying sequences not known by any published references to beassociated with a chromosomal disorder or disease. These clones areindicated in FIGS. 1-24 by left-pointing arrows, and may be consideredto be “non-reactive” or “backbone” clones, which act as positivehybridization controls, i.e., provide portions of the chromosome for anychromosome of interest that will hybridize to nucleic acid from a testsubject. The term, “non-reactive” means that the nucleic acid generallyhybridizes to a full extent to a genomic nucleic acid from any testsubject, i.e., and is “non-reactive” because it does not give a false“positive” diagnosis of a chromosomal disorder. Because the non-reactiveor backbone clones are positive controls for hybridization, they aretherefore expected to be non-reactive with a test sample for detectionof a chromosomal disorder.

A calibration spot may be a mixture of nucleic acids from backboneclones, for any one chromosome, or for all of the chromosomes in thehuman genome or genome of any other organism. An exemplary calibrationspot may comprise a mixture of nucleic acids from backbone clones, forexample, from about 10, from about 20, from about 40, or from about 80backbone or non-reactive clones. An exemplary calibration spot contains72 non-reactive backbone clones, selected to represent each of the setof autosomes and sex chromosomes. An alternative calibration spotcontains nucleic acid from an unrelated heterologous species, such as afish or amphibian, for purposes of standardizing hybridization, in whichcase an internal control carrying a recognizable label can be added or“spiked” into each hybridization mixture.

Representation of each chromosome is made by calculating ratios oflabels in each of the two double dye-labeled hybridizations (dye swap)and relative amounts are plotted graphically as a function of distanceof each cloned chromosomal portion from the p terminus conventionallyshown on the left. By convention, one of the two double labeledmaterials is plotted in a consistent line format (e.g., double line),and the other in a different color line format (e.g., solid line), suchthat deletion of a portion of nucleic acid in a test subject isdisplayed using a double line above the 1.0 ratio line (see FIGS.25A-25F and FIGS. 26A-26F), and an insertion such as an amplification isplotted as solid line above the 1.0 ratio line.

Analyses shown herein have made the unexpected finding that nucleic acidsequences in subjects corresponding to clones carrying nucleotidesequences closely linked to telomeres have a greater extent ofassociation with chromosomal disorders, while clones carryingcentromere-linked sequences have the least extent of chromosomaldisorders. A calibration spot that is a mixture of exclusivelycentromere-linked nucleic acids, or substantially havingcentromere-linked nucleic acids, provides a strong positive control thatis useful for one exemplary calibration spot as described herein.

In addition, the arrays provided herein as shown in drawings andexamples herein, include cloned nucleic acids from portions of eachchromosome that are not associated with any known chromosomal disorders,so that representations of a chromosome of a test subject's DNA isfacilitated, and a chromosomal disorder on a given chromosome is morereadily distinguished from normal portions of that chromosome.

Nucleic Acids and Detectable Moieties: Incorporating Labels and ScanningArrays

In making and using the nucleic acid compilations, or sets, libraries orcollections of the invention and arrays and practicing the methods ofthe invention, nucleic acids associated with a detectable label can bemade and used and incorporated into the compositions of the invention.The detectable label can be incorporated into, associated with orconjugated to a nucleic acid. Any detectable moiety can be used. Theassociation with the detectable moiety can be covalent or non-covalent.In another aspect, the array-immobilized nucleic acids and samplenucleic acids are differentially detectable, e.g., they have differentlabels and emit difference signals.

Useful labels include, e.g., ³²P, ³⁵S, ³H, ¹⁴C, ¹²⁵I, ¹³¹I; fluorescentdyes (e.g., Cy5™, Cy3™, FITC, rhodamine, lanthanide phosphors, Texasred), electron-dense reagents (e.g. gold), enzymes, e.g., as commonlyused in an ELISA (e.g., horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), colorimetric labels (e.g. colloidalgold), magnetic labels (e.g. Dynabeads™), biotin, dioxigenin, or haptensand proteins for which antisera or monoclonal antibodies are available.The label can be directly incorporated into the nucleic acid to bedetected, or it can be attached to a probe or antibody that hybridizesor binds to the target. A peptide can be made detectable byincorporating (e.g., into a nucleoside base) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, transcriptionalactivator polypeptide, metal binding domains, epitope tags). Label canbe attached by spacer arms of various lengths to reduce potential sterichindrance or impact on other useful or desired properties. See, e.g.,Mansfield (1995) Mol Cell Probes 9:145-156. In array-based CGH, fluorscan be paired together; for example, one fluor labeling the control(e.g., the “nucleic acid of “known, or normal, karyotype”) and anotherfluor the test nucleic acid (e.g., from a chorionic villus sample or acancer cell sample). Exemplary pairs are: rhodamine and fluorescein(see, e.g., DeRisi (1996) Nature Genetics 14:458-460);lissamine-conjugated nucleic acid analogs and fluorescein-conjugatednucleotide analogs (see, e.g., Shalon (1996) supra); Spectrum Red™ andSpectrum Green™ (Vysis, Downers Grove, IL.); Cy3™ and Cy5™. Cy3™ andCy5™ can be used together; both are fluorescent cyanine dyes produced byAmersham Life Sciences (Arlington Heights, IL.). Cyanine and relateddyes, such as merocyanine, styryl and oxonol dyes, are particularlystrongly light-absorbing and highly luminescent, see, e.g., U.S. Pat.Nos. 4,337,063; 4,404,289; 6,048,982.

Other fluorescent nucleotide analogs can be used, see, e.g., Jameson(1997) Methods Enzymol. 278:363-390; Zhu (1994) Nucleic Acids Res.22:3418-3422. U.S. Pat. Nos. 5,652,099 and 6,268,132 also describenucleoside analogs for incorporation into nucleic acids, e.g., DNAand/or RNA, or oligonucleotides, via either enzymatic or chemicalsynthesis to produce fluorescent oligonucleotides. U.S. Pat. No.5,135,717 describes phthalocyanine and tetrabenztriazaporphyrin reagentsfor use as fluorescent labels.

Detectable moieties can be incorporated into sample genomic nucleic acidand, if desired, any member of the compilation of nucleic acids orarray-immobilized nucleic acids, by covalent or non-covalent means,e.g., by transcription, such as by random-primer labeling using Klenowpolymerase, or “nick translation,” or, amplification, or equivalent. Forexample, in one aspect, a nucleoside base is conjugated to a detectablemoiety, such as a fluorescent dye, e.g., Cy3™ or Cy5™, and thenincorporated into a sample genomic nucleic acid. Samples of genomic DNAcan be incorporated with Cy3™- or Cy5™-dCTP conjugates mixed withunlabeled dCTP. Cy5™ is typically excited by the 633 nm line of HeNelaser, and emission is collected at 680 nm. See also, e.g., Bartosiewicz(2000) Archives of Biochem. Biophysics 376:66-73; Schena (1996) Proc.Natl. Acad. Sci. USA 93:10614-10619; Pinkel (1998) Nature Genetics20:207-211; Pollack (1999) Nature Genetics 23:41-46.

In another aspect, when using PCR or nick translation to label nucleicacids, modified nucleotides synthesized by coupling allylamine-dUTP tothe succinimidyl-ester derivatives of the fluorescent dyes or haptenes(such as biotin or digoxigenin) are used; this method allows custompreparation of most common fluorescent nucleotides, see, e.g., Henegariu(2000) Nat. Biotechnol. 18:345-348.

In the nucleic acid compilations, libraries, sets, or collections, orarrays and methods of the invention, labeling with a detectablecomposition (labeling with a detectable moiety) also can include anucleic acid attached to another biological molecule, such as a nucleicacid, e.g., a nucleic acid in the form of a stem-loop structure as a“molecular beacon” or an “aptamer beacon.” Molecular beacons asdetectable moieties are well known in the art; for example, Sokol (1998)Proc. Natl. Acad. Sci. USA 95:11538-11543, synthesized “molecularbeacon” reporter oligodeoxynucleotides with matched fluorescent donorand acceptor chromophores on their 5′ and 3′ ends. In the absence of acomplementary nucleic acid strand, the molecular beacon remains in astem-loop conformation where fluorescence resonance energy transferprevents signal emission. On hybridization with a complementarysequence, the stem-loop structure opens increasing the physical distancebetween the donor and acceptor moieties thereby reducing fluorescenceresonance energy transfer and allowing a detectable signal to be emittedwhen the beacon is excited by light of the appropriate wavelength. Seealso, e.g., Antony (2001) Biochemistry 40:9387-9395, describing amolecular beacon comprised of a G-rich 18-mer triplex formingoligodeoxyribonucleotide. See also U.S. Pat. Nos. 6,277,581 and6,235,504.

Aptamer beacons are similar to molecular beacons; see, e.g., Hamaguchi(2001) Anal. Biochem. 294:126-131; Poddar (2001) Mol. Cell. Probes15:161-167; Kaboev (2000) Nucleic Acids Res. 28:E94. Aptamer beacons canadopt two or more conformations, one of which allows ligand binding. Afluorescence-quenching pair is used to report changes in conformationinduced by ligand binding. See also, e.g., Yamamoto (2000) Genes Cells5:389-396; Smimov (2000) Biochemistry 39:1462-1468.

Detecting Dyes and Fluors

In addition to labeling nucleic acids with fluorescent dyes, theinvention can be practiced using any apparatus or methods to detect“detectable labels” of a sample nucleic acid, a member of thecompilation of nucleic acids, or an array-immobilized nucleic acid, or,any apparatus or methods to detect nucleic acids specifically hybridizedto each other. In one aspect, devices and methods for the simultaneousdetection of multiple fluorophores are used; they are well known in theart, see, e.g., U.S. Pat. Nos. 5,539,517; 6,049,380; 6,054,279;6,055,325; 6,294,331. Any known device or method, or variation thereof,can be used or adapted to practice the methods of the invention,including array reading or “scanning” devices, such as scanning andanalyzing multicolor fluorescence images; see, e.g., U.S. Pat. Nos.6,294,331; 6,261,776; 6,252,664; 6,191,425; 6,143,495; 6,140,044;6,066,459; 5,943,129; 5,922,617; 5,880,473; 5,846,708; 5,790,727; and,the patents cited in the discussion of arrays, herein. See alsopublished U.S. patent applications Nos. 20010018514; 20010007747;published international patent applications Nos. WO0146467 A; WO9960163A; WO0009650 A; WO0026412 A; WO0042222 A; WO0047600 A; WO0101144 A.

For example a spectrograph can image an emission spectrum onto atwo-dimensional array of light detectors; a full spectrally resolvedimage of the array is thus obtained. Photophysics of the fluorophore,e.g., fluorescence quantum yield and photodestruction yield, and thesensitivity of the detector are read time parameters for anoligonucleotide array. With sufficient laser power and use of Cy5™and/or Cy3™, which have lower photodestruction yields an array can beread in less than 5 seconds.

When using two or more fluors together (e.g., as in a CGH), such as Cy3™and Cy5™, it is necessary to create a composite image of all the fluors.To acquire the two or more images, the array can be scanned eithersimultaneously or sequentially. Charge-coupled devices, or CCDs, areused in microarray scanning systems, including practicing the methods ofthe invention. Thus, CCDs used in the methods of the invention can scanand analyze multicolor fluorescence images.

Color discrimination can also be based on 3-color CCD video images;these can be performed by measuring hue values. Hue values areintroduced to specify colors numerically. Calculation is based onintensities of red, green and blue light (RGB) as recorded by theseparate channels of the camera. The formulation used for transformingthe RGB values into hue, however, simplifies the data and does not makereference to the true physical properties of light. Alternatively,spectral imaging can be used; it analyzes light as the intensity perwavelength, which is the only quantity by which to describe the color oflight correctly. In addition, spectral imaging can provide spatial data,because it contains spectral information for every pixel in the image.Alternatively, a spectral image can be made using brightfieldmicroscopy, see, e.g., U.S. Pat. No. 6,294,331.

Data Analysis

The methods of the invention further comprise data analysis, which caninclude the steps of determining, e.g., fluorescent intensity as afunction of substrate position, removing “outliers” (data deviating froma predetermined statistical distribution), or calculating the relativebinding affinity of the targets from the remaining data. The resultingdata can be displayed as an image with color in each region varyingaccording to the light emission or binding affinity between targets andprobes. See, e.g., U.S. Pat. Nos. 5,324,633; 5,863,504; 6,045,996. Theinvention can also incorporate a device for detecting a labeled markeron a sample located on a support, see, e.g., U.S. Pat. No. 5,578,832.

Sources of Genomic Nucleic Acid

The invention provides methods of detecting a genetic mosaicism in anysample comprising nucleic acid, such as a cell population or tissue orfluid sample, by performing an array-based comparative genomichybridization (CGH). The nucleic acid can be derived from (e.g.,isolated from, amplified from, cloned from) genomic DNA. The genomic DNAcan be from any source.

In one aspect, the cell, tissue or fluid sample from which the nucleicacid sample is prepared is taken from a patient suspected of having apathology or a condition associated with genetic defects. The causality,diagnosis or prognosis of the pathology or condition may be associatedwith genetic defects, e.g., with genomic nucleic acid basesubstitutions, amplifications, deletions and/or translocations. Thecell, tissue or fluid can be from, e.g., amniotic samples, chorionicvillus samples (CVS), serum, blood, chord blood or urine samples, CSF orbone marrow aspirations, fecal samples, saliva, tears, tissue andsurgical biopsies, needle or punch biopsies, and the like.

Methods of isolating cell, tissue or fluid samples are well known tothose of skill in the art and include, but are not limited to,aspirations, tissue sections, drawing of blood or other fluids, surgicalor needle biopsies, and the like. A “clinical sample” derived from apatient includes frozen sections or paraffin sections taken forhistological purposes. The sample can also be derived from supernatants(of cell cultures), lysates of cells, cells from tissue culture in whichit may be desirable to detect levels of mosaicisms, includingchromosomal abnormalities and copy numbers.

Making Nucleic Acid Arrays

Making BAC Microarrays

Bacterial strains carrying BAC clones having cloned insert DNA ofobserved sizes greater than about fifty kilobases (50 kb), and up toabout 300 kb, are grown in Terrific Broth medium. Cells having BACclones with larger inserts, e.g., clones >300 kb, and smaller inserts,about 1 to 20 kb, may also be used. DNA is prepared in one embodiment bya modified alkaline lysis protocol (see, e.g., Sambrook), however, anyprotocol for purifying DNA is within the scope of the method. The DNA islabeled, as described below.

The DNA is then chemically modified as described by U.S. Pat. No.6,048,695. The modified DNA is then dissolved in proper buffer andprinted directly on clean glass surfaces as described by U.S. Pat. No.6,048,695. Usually multiple spots are printed for each clone.

Nucleic Acid Labeling and Fragmentation

A standard random priming method is used to label genomic DNA before itsattachment to the array, see, e.g., Sambrook. Sample nucleic acid isalso similarly labeled. Cy3™ or Cy5™ labeled nucleotides aresupplemented together with corresponding unlabeled nucleotides at amolar ratio ranging from 0.0 to about 6 (unlabeled nucleotide to labelednucleotides). Labeling is carried out at 37° C. for 2 to 10 hours. Afterlabeling the reaction mix is heated up to 95° C. to 100° C. for 3 to 5minutes to inactivate the polymerase and denature the newly generated,labeled “probe” nucleic acid from the template.

The heated sample is then chilled on ice for 5 minutes. “Calibrated”DNase (DNA endonuclease) enzyme is added to fragment the labeledtemplate (generated by random priming). “Trace” amounts of DNase isadded (final concentration was 0.2 to 2 ng/ml; incubation time 15 to 30minutes) to digest/fragment the labeled nucleic acid to segments ofabout 30 to about 100 bases in size. Alternatively, DNA is fragmented bysonication.

Nucleic acids are alternatively fragmented by sonication. Sonicationprotocols include establishing standard power levels and times ofsonication, to obtain fragments of desired length.

Hybridization of Nucleic Acid Samples to Arrays

The examples set forth herein provide exemplary methods for pretreatingnucleic acid samples and hybridizing these samples to arrays. Thisexemplary hybridization protocol can be used to determine if a nucleicacid segment, such as a genomic clone, is within the scope of theinvention (e.g., is a member of a compilation, library, clone set of theinvention).

Pretreatment of Sample DNA

Random prime labeling of large sized DNA samples, such as genomic DNA,can be more efficient if the DNA sample is first digested or otherwisetreated to produce smaller fragments of more uniform size and mobilityin solution. For every test sample to be analyzed, four digests ofgenomic DNA were performed: two of the test sample and two of anappropriate reference or control sample.

-   -   1. Restriction enzyme digest of genomic DNA: on ice, pipet the        following into an autoclaved microcentrifuge tube:    -   DNA× μl for 1 μg    -   React 3 10× Buffer 5 μl    -   Eco R1 2 μl (20units)    -   Water (orange vial) μl to a final volume of 50 μl    -   2. After addition of the enzyme and DNA, mix briefly by        vortexing and recollect samples by brief centrifugation.    -   3. Incubate samples overnight (16 hours) at 37° C.    -   4. Determine the completion of the reaction by removing a 5 μl        aliquot from the reaction mix, and analyzing the aliquot by        agarose gel electrophoresis (0.8% agarose).    -   If the digestion is complete, stop the reaction by incubating in        a heating block at 72° C. for 10 minutes. It is recommended to        fill the wells of the heating block with water approximately 15        minutes before denaturing the samples so that the tubes are in        contact with water at 72° C.    -   5. Re-purify the digested DNA sample (either by        phenol/chloroform extraction/EtOH precipitation or a suitable        commercially available ‘post-enzyme digestion/PCR clean-up kit’        such as Zymo Research's DNA Clean and Concentrator TM-5 Cat No.        D4005). Note: It is recommended to requantifying the DNA samples        at this juncture to ensure that equitable amounts of the test        and reference samples will be labeled in the following step.    -   At least 500 ng of digested DNA of each sample were used for        labeling.    -   Genomic DNA samples adequately digested with a four base pair        (4-bp) cutter restriction enzyme, such as EcoR1, should produce        a relatively homogenous smear extending from 20 kb to        approximately 600 bp.        Differential Labeling of DNA with Cy3-dCTP and Cy5-dCTP

The objective in this step is to label the test and reference sampleswith both Cy-3 and Cy-5 to facilitate the co-hybridization between theCy-3 labeled test and Cy-5 labeled reference samples, and conversely theCy-5 labeled test and Cy-3 labeled reference samples.

-   -   1. To the re-purified DNA samples, add sterile water to bring        the total volume to 25 μl. Then add 20 μl of 2.5× random        primer/reaction buffer mix (e.g., from Gibco/BRL's BIOPRIME™        labeling kit).    -   2. Mix the samples well and then boil for 5 minutes.    -   3. Immediately place the samples on ice and allow to sit for 5        minutes.    -   4. On ice, add 2.5 μl of SPECTRAL LABELING BUFFER, for use with        SPECTRAL CHIP™ (Spectral Genomics, Houston Tex.) to each sample.    -   5. Add 1.5 μl Cy5-dCTP or Cy3-dCTP to the respective test and        reference DNA samples (1 mM stocks).    -   6. Finally, add 1 μl Klenow Fragment (from the Gibco/BRL        BIOPRIME™ labeling kit) to the samples, mix the sample well by        tapping, and re-collect by brief centrifugation.    -   7. Incubate the sample at 37° C for 1½-2 hours. Place the        samples on ice and determine the probe size distribution by        removing a 5 μl aliquot from the reaction mix, and analyzing the        aliquot by agarose gel electrophoresis (0.8% agarose). Note:        Optimally, the majority of the probe should range in size        between 100-500 bp.    -   8. Stop the reaction by adding 5 μl 0.5 M EDTA pH8.0 and        incubating in a heating block at 72° C. for 10 minutes. Place        the samples on ice. The samples can now be used to proceed with        hybridization or can be stored at −20° C. until required.    -   Optimally the majority of the probe should range in size between        100-500 bp.        Hybridizing Labeled DNA to the Array

At this juncture, there should be four tubes, which should correspond tothe Cy-3 and Cy-5 labeled test samples and the Cy-3, and Cy-5 labeledreference samples.

-   -   1. Combine the Cy3-labeled test DNA sample with the Cy5-labeled        reference sample and, conversely, the Cy5-labeled test DNA        sample with the Cy3-labeled reference sample. Add 45 μl of        SPECTRAL HYBRIDIZATION BUFFER I, for use with SPECTRAL CHIP™        (Spectral Genomics, Houston Tex.) to each of the two tubes.    -   2. Precipitate the two samples by adding 11.3 μl of 5MNaCl and        101 μl of room temperature isopropanol. Mix the samples well and        incubate in the dark at room temperature for 10-15 minutes.    -   3. Centrifuge the samples at full speed (10,000 g) for 10        minutes.    -   4. Aspirate the supernatant, avoiding the pellet. Note: The        pellets should have a purplish hue, indicating that there are        expected amounts of Cy3 and Cy5 labeled DNA. Too pink or too        blue a sample, suggests that the corresponding genomic DNA was        not appropriately labeled.    -   5. Rinse the pellets with 500 μl of 70% ethanol and allow the        pellets to air-dry briefly in the dark at room temperature.    -   6. Add 10 μl of sterile water (orange vial) to the pellets. Let        stand at room temperature for 5 minutes and then thoroughly        resuspend. After ensuring that the pellets are completely        resuspended, add 30 μl of SPECTRAL HYBRIDIZATION BUFFER II, for        use with SPECTRAL CHIP™ (Spectral Genomics, Houston Tex.) and        mix well by repeated pipetting.    -   7. Denature the samples by incubating in a water bath at 72° C.        for 10 minutes. Note: Alternatively, the sample can be denatured        in a heating block set at 72° C. We recommend filling the wells        of the heating block with water approximately 15 minutes before        denaturing the samples so that the tubes are in contact with        water at 72° C.    -   8. After the denaturation of the samples, immediately place the        tubes on ice for 5 minutes.    -   9. Incubate the samples at 37° C. for 30 minutes.    -   10. Pipette the sample onto the center of the array and cover        with a 22×60 cover slip to spread it out. Note: It is imperative        that the entire array is covered and that air bubbles are        avoided.    -   11. Place the slide in a hybridization chamber. If a microarray        hybridization chamber is used, then add 10 μl of 2×SSC, 50%        formamide to either side of the chamber. (H₂O works just as        well).    -   12. Close the chamber and wrap with aluminum foil. Put the        chambers in a Kapak Pouch with wet paper and heat seal the bag.        Put the bag in a 37° C. incubator for 16 hours. Note: We        recommend using a shaking platform incubator to facilitate and        maintain even distribution of the probe on the slide.        Post Hybridization Washes

While Coplin jars can be used in the post-hybridization washes, it isrecommended to wash each slide in individual Petri dishes in a shakingplatform incubator.

-   -   1. Pre-warm the following solutions at 50° C. in individual        Petri dishes:    -   2×SSC, 50% deionized Formamide    -   2×SSC, 0.1% NP-40    -   0.2×SSC    -   2. Soak the slide in 2×SSC, 0.5% SDS briefly at room temperature        and gently slide off the cover slip using a pair of clean        forceps. Avoid peeling off the cover slip by force.        (Alternatively, 2×SSC can be used)    -   3. Using a pair of forceps, transfer the slide to pre-warmed        2×SSC, 50% Formamide. Wash the slides by incubating in the        shaking incubator at 50° C. for 20 minutes.    -   4. Repeat step 3 using pre-warmed 2×SSC, 0.1% NP-40.    -   5. Repeat step 3 using pre-warmed 0.2×SSC for 10 minutes.    -   6. Briefly rinse the slides with distilled deionized water. This        last wash greatly reduces background fluorescence but should not        exceed 10 seconds.    -   7. Immediately dry the slides under forced air. Do not air dry        the slides. The slides are now ready for scanning.

EXAMPLES Example 1 Analysis of Chromosomal Disorders on Human Chromosome1

In FIG. 1 is seen an ideogram of chromsome 1. On the right side of theideogram and in the table are listed three BAC clones carrying nucleicacid sequences corresponding to a syndrom known as 1p36, and six clonesassociated with a syndrome known as 1q44, which are named for knownchromosomal disorders that are associated with known areas of chromosome1.

Also shown are nine clones carrying portions of chromosome 1 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 1, which is indicated by the constriction inthe ideogram between 1p11 and 1q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 1 that containthese loci on chromosome 1, and the slides further had two copies ofeach of the microarrays.

Example 2 Analysis of Chromosomal Disorders on Human Chromosome 2

In FIG. 2 is seen an ideogram of chromsome 2. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to a syndrom known as 2p25.3, and fourclones associated with a syndrome known as 2q37.3, which are named knownchromosomal disorders that are associated with known areas of chromosome2.

Also shown are seven clones carrying portions of chromosome 2 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 2, which is indicated by the constriction inthe ideogram between 2p11 and 2q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 2 that containthese loci on chromosome 2, and the slides further had two copies ofeach of the microarrays.

Example 3 Analysis of Chromosomal Disorders on Human Chromosome 3

In FIG. 3 is seen an ideogram of chromsome 3. On the right side of theideogram and in the table are listed four BAC clones carrying nucleicacid sequences corresponding to a syndrome known as 3p26, and fiveclones associated with a syndrome known as 3q25, and two clonesassociated with 3q29, which are named for known chromosomal disordersthat are associated with known areas of chromosome 3.

Also shown are seven clones carrying portions of chromosome 3 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 3, which is indicated by the constriction inthe ideogram between 3p11 and 3q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 3 that containthese loci on chromosome 3, and the slides further had two copies ofeach of the microarrays.

Example 4 Analysis of Chromosomal Disorders on Human Chromosome 4

In FIG. 4 is seen an ideogram of chromsome 4. On the right side of theideogram and in the table are listed ten BAC clones carrying nucleicacid sequences corresponding to locus 4p16, some of which are associatedwith a syndrome known as Wolf-Hirschhom syndrome, and sev clonesassociated with a syndrome known as 3q25, and two clones associated with4q35, which are named for known chromosomal disorders that areassociated with known areas of chromosome 4.

Also shown are seven clones carrying portions of chromosome 4 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 4, which is indicated by the constriction inthe ideogram between 4p11 and 4q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 4 that containthese loci on chromosome 4, and the slides further had two copies ofeach of the microarrays.

Example 5 Analysis of Chromosomal Disorders on Human Chromosome 5

In FIG. 5 is seen an ideogram of chromsome 5. On the right side of theideogram and in the table are listed ten BAC clones carrying nucleicacid sequences corresponding to locus 5p15, some of which are associatedwith a syndrome known as Cri-du-Chat syndrome, and seven clonesassociated with a syndrome at locus 5q35 known as Sotos Syndrom+C92s,which are known chromosomal disorders that are associated with knownareas of chromosome 5.

Also shown are seven clones carrying portions of chromosome 5 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 5, which is indicated by the constriction inthe ideogram between 5p11 and 5q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 5 that containthese loci on chromosome 5, and the slides further had two copies ofeach of the microarrays.

Example 6 Analysis of Chromosomal Disorders on Human Chromosome 6

In FIG. 6 is seen an ideogram of chromsome 6. On the right side of theideogram and in the table are listed ten BAC clones carrying nucleicacid sequences corresponding to locus 6p25, which are associated with asyndrome, and three clones associated with a syndrome at locus 6q27,which are known chromosomal disorders that are associated with knownareas of chromosome 6.

Also shown are four clones carrying portions of chromosome 6 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 6, which is indicated by the constriction inthe ideogram between 6p11 and 6q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 6 that containthese loci on chromosome 6, and the slides further had two copies ofeach of the microarrays.

Example 7 Analysis of Chromosomal Disorders on Human Chromosome 7

In FIG. 7 is seen an ideogram of chromsome 7. On the right side of theideogram and in the table are listed five BAC clones carrying nucleicacid sequences corresponding to locus 7p22.3 and 7p21, three of whichare associated with a syndrome known as Saethre-Chotzen Syndrom, andthree clones associated with a syndrome at locus 7p13 known as Greigcephelopolysyndactyly syndrome, and 13 clones associated with 7q11.23associated with Williams-Beuren syndrom, and four clones associated withlocus 7q36.3, which are known chromosomal disorders that are associatedwith known areas of chromosome 7.

Also shown are four clones carrying portions of chromosome 7 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, one of these clones are closely linked to thecentromere of chromosome 7, which is indicated by the constriction inthe ideogram between 7p11 and 7q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 7 that containthese loci on chromosome 7, and the slides further had two copies ofeach of the microarrays.

Example 8 Analysis of Chromosomal Disorders on Human Chromosome 8

In FIG. 8 is seen an ideogram of chromsome 8. On the right side of theideogram and in the table are listed eight BAC clones carrying nucleicacid sequences corresponding to locus 8p22, which are associated with asyndrome known as Kabuki syndrome, and four clones associated with asyndrome at locus 8q24.12 associated with Trichorhinophalangeal Syndrometype 1 (TRPS1), and three clones associated with 8q24.12 associated withExotoses, multiple, type 1 (EXT1) syndrome, and three clones associatedwith locus 8q24.3, which are known chromosomal disorders that areassociated with known areas of chromosome 8.

Also shown are seven clones carrying portions of chromosome 8 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 8, which is indicated by the constriction inthe ideogram between 8p11 and 8q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 8 that containthese loci on chromosome 8, and the slides further had two copies ofeach of the microarrays.

Example 9 Analysis of Chromosomal Disorders on Human Chromosome 9

In FIG. 9 is seen an ideogram of chromsome 9. On the right side of theideogram and in the table are listed three BAC clones carrying nucleicacid sequences corresponding to locus 9p24.3, and three clonesassociated with a syndrome at locus 9q34.3, which are known chromosomaldisorders that are associated with known areas of chromosome 9.

Also shown are five clones carrying portions of chromosome 9 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, some of these clones are closely linked to thecentromere of chromosome 9, which is indicated by the constriction inthe ideogram between 9p11 and 9q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 9 that containthese loci on chromosome 9, and the slides further had two copies ofeach of the microarrays.

Example 10 Analysis of Chromosomal Disorders on Human Chromosome 10

In FIG. 10 is seen an ideogram of chromsome 10. On the right side of theideogram and in the table are listed three BAC clones carrying nucleicacid sequences corresponding to locus 10p15.3, and three clonesassociated with a syndrome at locus 10p14 known as HDR syndrome, andfive clones associated with a syndrome between loci 10p14 and 10p13known as DiGeorge Syndrome/velocardiofacial Syndrome complex-2 (DGS2 orDGCR2), and one clone at locus 10p12.31 known as Nebulette (NEBL), andone clone at locus 10p12.33 associated with DGS2 or DGCR2 syndrome, andsix lones associated with locus 10q24 known as Split foot-Split Handsyndrome, and three clones at locus 10q26.3, which are known chromosomaldisorders that are associated with known areas of chromosome 10.

Also shown are four clones carrying portions of chromosome 10 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, two of these clones are closely linked to thecentromere of chromosome 10, which is indicated by the constriction inthe ideogram between 10p11 and 10q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 10 that containthese loci on chromosome 10, and the slides further had two copies ofeach of the microarrays.

Example 11 Analysis of Chromosomal Disorders on Human Chromosome 11

In FIG. 11 is seen an ideogram of chromsome 11. On the right side of theideogram and in the table are listed four BAC clones carrying nucleicacid sequences corresponding to locus 11p15.5, two of which areassociated with Beckwith-Wiedemann Syndrome (BWS), and one cloneassociated with WAGR syndrome at locus 11p13, and one clone associatedwith Wilm's Tumor syndrome at locus 11p13, and four clones associatedwith a syndrome at locus 11p11.2 known as Potocki-Shaffer Syndrome(PSS), and three clones at locus 11q25, which are known chromosomaldisorders that are associated with known areas of chromosome 11.

Also shown are four clones carrying portions of chromosome 11 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, one of these clones isclosely linked to the centromereof chromosome 11, which is indicated by the constriction in the ideogrambetween 11p11 and 11q11. Microarrays were prepared on glass slideshaving DNA from the listed BAC clones in FIG. 11 that contain these locion chromosome 11, and the slides further had two copies of each of themicroarrays.

Example 12 Analysis of Chromosomal Disorders on Human Chromosome 12

In FIG. 12 is seen an ideogram of chromsome 12. On the right side of theideogram and in the table are listed four BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome between loci12p13.33 and 12p13.32, and three clones at locus 12q24.33, which areknown chromosomal disorders that are associated with known areas ofchromosome 12.

Also shown are four clones carrying portions of chromosome 12 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, one of these clones is closely linked to thecentromere of chromosome 12, which is indicated by the constriction inthe ideogram between 12p11 and 12q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 12 that containthese loci on chromosome 12, and the slides further had two copies ofeach of the microarrays.

Example 13 Analysis of Chromosomal Disorders on Human Chromosome 13

In FIG. 13 is seen an ideogram of chromsome 13. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus13q14 and associated with retinoblastoma 1 (RB 1), and four clonesassociated with loci between 13q32 and 13q34, which are knownchromosomal disorders that are associated with known areas of chromosome13.

Also shown are two clones carrying portions of chromosome 13 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, one of these clones is closely linked to thecentromere of chromosome 13, which is indicated by the constriction inthe ideogram between 13p11 and 13q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 13 that containthese loci on chromosome 13, and the slides further had two copies ofeach of the microarrays.

Example 14 Analysis of Chromosomal Disorders on Human Chromosome 14

In FIG. 14 is seen an ideogram of chromsome 14. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus14q24.3 and associated with known chromosomal disorders that areassociated with known areas of chromosome 14.

Also shown are four clones carrying portions of chromosome 14 that arenot associated with known chromosomal disorders (leftward pointingarrows). Further, one of these clones is closely linked to thecentromere of chromosome 14, which is indicated by the constriction inthe ideogram between 14p11 and 14q11. Microarrays were prepared on glassslides having DNA from the listed BAC clones in FIG. 14 that containthese loci on chromosome 14, and the slides further had two copies ofeach of the microarrays.

Example 15 Analysis of Chromosomal Disorders on Human Chromosome 15

In FIG. 15 is seen an ideogram of chromsome 15. On the right side of theideogram and in the table are listed six BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus15p11.2 and associated with known chromosomal disorder Prader-WilliSyndrome (PWS)/Angelman Region (AR), and three clones at locus 15p14,and one clone at locus 15q11.2 and associated with known chromosomaldisorder Prader-Willi Syndrome (PWS)/Angelman Region (AR), and oneadditional clone at 15q14, and three clones at 15q34, that areassociated with known areas of chromosome 14.

Also shown are three clones carrying portions of chromosome 15 that arenot associated with known chromosomal disorders (leftward pointingarrows). Microarrays were prepared on glass slides having DNA from thelisted BAC clones in FIG. 15 that contain these loci on chromosome 15,and the slides further had two copies of each of the microarrays.

Example 16 Analysis of Chromosomal Disorders on Human Chromosome 16

In FIG. 16 is seen an ideogram of chromsome 16. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus16p13.3, and three more clones associated with this locus and alsoassociated with known chromosomal disorder Tuberous sclerosis-2(TSC2)/Polycystic kidney disease adult type (PKD1), and four clones atlocus 16p13.3 associated with Rubinstein-Taybi Syndrome (RTS), and threeclones carrying portions of DNA from between loci 16q24.2 and 16q24.3,associated with known chromosomal disorders associated with known areasof chromosome 16.

Also shown are five clones carrying portions of chromosome 16 that arenot associated with known chromosomal disorders (leftward pointingarrows). Some of these are associated with regions of the chromosomehear the centromer. Microarrays were prepared on glass slides having DNAfrom the listed BAC clones in FIG. 16 that contain these loci onchromosome 16, and the slides further had two copies of each of themicroarrays.

Example 17 Analysis of Chromosomal Disorders on Human Chromosome 17

In FIG. 17 is seen an ideogram of chromsome 17. On the right side of theideogram and in the table are listed six BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus17p13.3, four of which associated with known chromosomal disorderMiller-Dieker Syndrome, and 16.clones at locus 17p11.2, seven of whichare associated with Charcot-Marie-Tooth disease, type 1A (CMT1A), andsix clones of which are associated with Smith-Magenis Syndrome (SMS).The ideogram and table further show three additional clones at 17q11.2associated with Neurofibromatosis, type 1 (NF 1), and fove clones at17q25.3, that are associated with known areas of chromosome 14 and knownchromosomal disorders.

Also shown are three clones carrying portions of chromosome 17 that arenot associated with known chromosomal disorders (leftward pointingarrows). Microarrays were prepared on glass slides having DNA from thelisted BAC clones in FIG. 17 that contain these loci on chromosome 17,and the slides further had two copies of each of the microarrays.

Example 18 Analysis of Chromosomal Disorders on Human Chromosome 18

In FIG. 18 is seen an ideogram of chromsome 18. On the right side of theideogram and in the table are listed six BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome between loci18p11.3 and 18p11.2, one clone at locus 18q12, and five clones betweenloci 18q21 and 18q23, which are associated with known areas ofchromosome 18 and known chromosomal disorders.

Also shown is one clone carrying a portion of chromosome 18 that is notassociated with known chromosomal disorders (leftward pointing arrows).Microarrays were prepared on glass slides having DNA from the listed BACclones in FIG. 18 that contain these loci on chromosome 18, and theslides further had two copies of each of the microarrays.

Example 19 Analysis of Chromosomal Disorders on Human Chromosome 19

In FIG. 19 is seen an ideogram of chromsome 19. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus19p13.3, and two clones for portions of the chromosome between loci19q13.4 and 19q13.43, which are associated with known areas ofchromosome 19 and known chromosomal disorders.

Also shown are two clones carrying portions of chromosome 19 that arenot associated with known chromosomal disorders (leftward pointingarrows). Microarrays were prepared on glass slides having DNA from thelisted BAC clones in FIG. 19 that contain these loci on chromosome 19,and the slides further had two copies of each of the microarrays.

Example 20 Analysis of Chromosomal Disorders on Human Chromosome 20

In FIG. 20 is seen an ideogram of chromsome 20. On the right side of theideogram and in the table are listed two BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome at locus20p13, and two clones for portions of the chromosome at locus 19q13.4and 20p12 that correspond to Alagille Syndrome (AGS), and three cloneswith portions of the choromosome from between loci 20q13.3 and 10q13.33,which are associated with known areas of chromosome 20 and knownchromosomal disorders.

Also shown are three clones carrying portions of chromosome 20 that arenot associated with known chromosomal disorders (leftward pointingarrows), one of which is closely linked to the centromere of chromosome20. Microarrays were prepared on glass slides having DNA from the listedBAC clones in FIG. 20 that contain these loci on chromosome 20, and theslides further had two copies of each of the microarrays.

Example 21 Analysis of Chromosomal Disorders on Human Chromosome 21

In FIG. 21 is seen an ideogram of chromsome 21. On the right side of theideogram and in the table are listed six BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome between loci21q21 and 21q22.3, one of which is associated with Down SyndromeCritical Region Gene 1 (DSCR1), and which are associated with knownareas of chromosome 21 and known chromosomal disorders, particularlyDown Syndrome.

Also shown is one clone carrying a portion of chromosome 21 that is notassociated with known chromosomal disorders (leftward pointing arrows),and which is closely linked to the centromere of chromosome 21.Microarrays were prepared on glass slides having DNA from the listed BACclones in FIG. 21 that contain these loci on chromosome 21, and theslides further had two copies of each of the microarrays.

Example 22 Analysis of Chromosomal Disorders on Human Chromosome 22

In FIG. 22 is seen an ideogram of chromsome 22. On the right side of theideogram and in the table are listed eleven BAC clones carrying nucleicacid sequences corresponding to portions of the chromosome between loci22q11.2 and 22q13.33, one of which is associated with DiGeorge SyndromeVelocardiofacial Syndrome (DGS/VCFS), one of which is associated withDiGeorge Syndrome Velocardiofacial Syndrome (DGS/VCFS)/T-box 1 (TBX1),and five of which are associated with DiGeorge Syndrome (DGS). Further,four clones contain portions of chromosome 22 at locus 22q13.33 and areassociated with chromosomal disorders at this site.

Also shown is one clone carrying a portion of chromosome 22 that is notassociated with known chromosomal disorders (leftward pointing arrows).Microarrays were prepared on glass slides having DNA from the listed BACclones in FIG. 22 that contain these loci on chromosome 22, and theslides further had two copies of each of the microarrays.

Example 23 Analysis of Chromosomal Disorders on Human Chromosome X

In FIG. 23A is seen an ideogram of chromsome X. On the right side of theideogram and in the table of FIG. 23B are listed 59 BAC clones carryingnucleic acid sequences corresponding to portions of the chromosomebetween loci Xp22.33 and Xq27.3. These clones are associated with alarge number of chromosomal disorders, including: Placental steroidsulfatase deficiency (STS) at locus Xp22.32; Kallmann Syndrome (KAL) atlocus Xp22.32; MLS syndrome deletion at loci Xp22.22 and locus Xp22.32;Glycerol Kinase deficiency at locus Xp21.3; Glycerol Kinasedeficiency/Adrenal hypoplasia, congenital (AHC) also at locus Xp21.3;Duchenne Muscular Dystrophy (DMD) from locus Xp21.3 to Xp21.2;Pelizaeus-Merzbacher Disease (PMD) at locus Xq22.2; and Fragile X mentalretardafion-1 (FMR1) at locus Xq27.3.

Also shown are three clones carrying portions of chromosome X that arenot associated with known chromosomal disorders (leftward pointingarrows), one of which is closely linked to the centromere. Microarrayswere prepared on glass slides having DNA from the listed BAC clones inFIGS. 23A-23B that contain these loci on chromosome X, and the slidesfurther had two copies of each of the microarrays.

Example 24 Analysis of Chromosomal Disorders on Human Chromosome Y

In FIG. 24 is seen an ideogram of chromsome Y. On the right side of theideogram and in the table are listed BAC clones carrying nucleic acidsequences corresponding to portions of the Y chromosome and associatedwith chromosomal disorders, most of which have a phenotype affectinggonadal development (associated with locus Yq3 or Yq21) or spermformation (Azoospermia factors 1, 2, and c, associated with locus Yq11).

Microarrays were prepared on glass slides having DNA from the listed BACclones in FIG. 24 that contain these loci on chromosome Y, and theslides further had two copies of each of the microarrays.

Example 26 Analysis of Test Sample Nucleic Acid with Telomere-LinkedClones for a Disease

FIGS. 25A-25F are representation of exemplary human chromosomes 1-6,using data obtained from dye swap analysis using the multiple singlesurface technique as described herein. For each chromosome indicated inthe upper right hand corner of the computer generated data, the pterminus is on the left, and the q terminus on the right. A lineparallel to the abscissa having a ratio of 1.0 indicates equalquantities of binding of test sample and reference sample nucleic acidsto the spot having the cloned nucleic acid. Comparison of the two setsof dye-swap data at each point is used to produce the two lines.Deviation from a ratio of 1.0 for both lines is associated withinsertion or deletion of genetic material.

The p terminus of chromosome 4 shown in FIGS. 25A-25F was found to havea substantial deviation from a ratio of 1.0. A typical software analysisindicates open squares connected by a double line above the 1.0 ratiofor a chromosomal disorder that indicates a deletion of material. Infact the sample nucleic acid is from a patient having Wolf-HirschhornSyndrome.

Example 27 Analysis of Test Sample Nucleic Acid Using Telomere- andCentromere-Linked Clones

Initially, only disease-associated and telomere-linked markersassociated with chromosomal disorders, for each chromosome, were used toform the array, and the data are shown in FIGS. 25A-25F.

The data in FIGS. 26A-26F form a representation of human chromosomes1-6. obtained by multiple array format dye swap analysis, using bothtelomere-linked cloned loci associated with chromosomal disorders, foreach chromosome, and control loci that are not associated with knownchromosomal disorders, including control loci that are linked to thecentromeres for each of the chromosomes. The test sample is from thesame patient as the data shown in Example 26.

For chromosome 4, it can be seen that the deletion is confined to the pterminus, and that the remainder of the chromosome appears to have thenormal complement of sequence material compared to the normal referencenucleic acid sample.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. A surface for detecting a genetic syndrome of a subject, the surfacecomprising a plurality of non-contiguous microarrays, wherein eachmicroarray comprises a plurality of cloned genomic nucleic acidsimmobilized on the surface at discrete and known spots, and eachmicroarray comprises a first set of spots having nucleic acids selectedto detect a genetic syndrome and a second set of spots having controlnucleic acids for at least part of a chromosome of the subject.
 2. Acombination comprising the surface according to claim 1, and a cover forat least one of the plurality of microarrays.
 3. The combinationaccording to claim 2, wherein the cover separates fluid of the at leastone microarray from other microarrays in the plurality of microarrays.4. The combination according to claim 3, wherein the surface is planar,and the cover is planar or arcuate in cross section.
 5. The surfaceaccording to claim 1, wherein the surface is selected from the groupconsisting of a metal, silicon, a polymer plastic, paper, ceramic,quartz, gallium arsenide, a metalloid, cellulose, cellulose acetate,nitrocellulose, and a glass.
 6. The surface according to claim 5,wherein the plastic is selected from the group consisting of nylon,polycarbonate, polyethylene, polystyrene, polytetrafluoroethylene,polypropylene, poly(4-methylbutene), polystyrene, latex,polymethacrylate, poly(ethylene terephthalate), rayon,polyvinylbutyrate, polyvinylidene difluoride, and combinations thereof.7. The surface according to claim 1, wherein the cloned genomic nucleicacids of the microarray comprise nucleic acids of a plurality ofchromosomes.
 8. The surface according to claim 7, wherein the pluralityof chromosomes comprises the genome of the subject.
 9. The surfaceaccording to claim 7, wherein the plurality of chromosomes comprisesautosomes.
 10. The surface according to claim 7, wherein the pluralityof chromosomes comprises at least one sex chromosome.
 11. The surfaceaccording to claim 1, wherein the cloned genomic nucleic acids of themicroarray comprise nucleic acids of a single chromosome.
 12. Thesurface according to claim 1, wherein the microarray comprises aplurality of cloned genomic nucleic acids selected to detect a geneticsyndrome and a plurality of cloned control genomic nucleic acids. 13.The surface according to claim 12, wherein the plurality of clonednucleic acids selected to detect a genetic syndrome are located on asingle chromosome.
 14. The surface according to claim 12, wherein theplurality of cloned nucleic acids selected to detect a genetic syndromeare located on a plurality of chromosomes.
 15. The surface according toclaim 12, wherein the plurality of cloned genomic nucleic acids selectedto detect a genetic syndrome and the plurality of cloned control genomicnucleic acids are distributed among a plurality of chromosomes of thesubject.
 16. The surface according to claim 1, wherein the microarrayfurther contains a plurality of cloned nucleic acids selected to detecta plurality of genetic syndromes.
 17. The surface according to claim 16,wherein the plurality is at least 5 genetic syndromes.
 18. The surfaceaccording to claim 16, wherein the plurality is at least 40 geneticsyndromes.
 19. The surface according to claim 16, wherein the pluralityis less than 1,000 genetic syndromes.
 20. The surface according to claim1, wherein the microarray further comprises at least one calibrationspot.
 21. The surface according to claim 20, wherein the calibrationspot comprises a mixture of the plurality of cloned genomic controlnucleic acids.
 22. The surface according to claim 20, wherein thecalibration spot comprises cloned genomic nucleic acids selected todetect a genetic syndrome and located on the chromosome closely linkedto centromere.
 23. The surface according to claim 1, wherein the surfacefurther comprises at least one fluid barrier located between at leasttwo of the microarrays, wherein the barrier separates a fluid above atleast one of the microarrays from fluid above at least anothermicroarray of the plurality.
 24. The surface according to claim 23,wherein the barrier is selected from an elevated structure ofhydrophobic composition contiguous with the surface and a hydrophobicstrip printed on the surface.
 25. The surface according to claim 23,wherein the elevated structure is a glass barrier.
 26. The surfaceaccording to claim 21, wherein the elevated structure comprises ahydrophobic polymer.
 27. The surface according to claim 21, wherein thehydrophobic strip is selected from the group of polyethylene, silicone,paraffin, and polytetrafluoroethylene.
 28. A kit comprising the surfaceof any of claims 1-27, a container, and instructions for use.
 29. Thekit according to claim 28, further comprising nucleic acid of areference subject.
 30. The kit according to claim 29, further comprisinga first detectable label and a second detectable label.